UNIVERSITY OF CALGARY. The Role of Integrin alpha1beta1 and Epidermal Growth Factor Receptor Signalling in Posttraumatic. Sung Yong Shin A THESIS

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1 UNIVERSITY OF CALGARY The Role of Integrin alpha1beta1 and Epidermal Growth Factor Receptor Signalling in Posttraumatic Osteoarthritis by Sung Yong Shin A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN KINESIOLOGY CALGARY, ALBERTA MAY, 2015 SUNG YONG SHIN 2015

2 Abstract Purpose: The purpose of this thesis was to investigate the role of integrin α1β1 in the progression of post-traumatic osteoarthritis (PTOA), and epidermal growth factor receptor (EGFR) signalling as the mechanism(s) by which integrin α1β1 might delay signs of PTOA. Methods: Surgery to destabilise the medial meniscus was performed on integrin α1-null and wildtype mice and the progression of PTOA was monitored for 12 weeks using microct, histology, and behavioural testing. The EGFR-inhibitor erlotinib was administered to a subset of mice. Results: Cartilage damage occurred four weeks earlier in α1-null compared to WT female mice. Independent of genotype, cartilage damage and bony signs of PTOA were lessened by erlotinib treatment in female mice. Conclusion: Integrin α1β1 protects against PTOA-induced cartilage degradation up to 8 weeks postsurgery partially via the dampening of EGFR signalling, in female mice. Furthermore, EGFR signalling aggravates the development of PTOA in female but not male mice. ii

3 Acknowledgements The journey I have taken through graduate school was a one of a kind experience, with a blend of woos and woes that helped me to grow. I am grateful to have had invaluable support from a handful of individuals who dedicated their time and effort to ensure the success of this project. I would like to express my most sincere appreciation to: My supervisor, Dr. Andrea Clark, for her enthusiasm, emotional support, and guidance, which helped me through the difficult times and motivated me to push my limits. My supervisory committee, Drs. Walter Herzog, and Steve Boyd, for their advice and support. Dr. Kevin Hildebrand for accepting to be an examiner of my defense as well as for the opportunities to shadow his clinical work. Dr. Tak Fung for his humour and statistical expertise. Dr. Ambra Pozzi for providing the integrin α1-null mice and for the knowledge and insight she has contributed to the project. Kevin Chapman for his expertise in behavioural testing. Carin Pihl and Dawn Martin for expert assistant in all animal procedures, Britta Jorgenson for microct expertise, Hakan Kadir and Charlie Shin for gavage assistance, and Hakan Kadir, Lisa Milo, Erica Floreani, and Dilene Mugenzi for data collection. My family for their unconditional support and faith in my abilities to succeed through this academic journey. iii

4 Dedication To my family iv

5 Table of Contents Abstract... ii Acknowledgements... iii Table of Contents...v List of Tables... viii List of Figures and Illustrations... ix List of Symbols, Abbreviations and Nomenclature... xvi Epigraph... xvii CHAPTER ONE: LITERATURE REVIEW Osteoarthritis Epidemiology Signs and Symptoms Risk Factors Systemic Local Treatments Nonpharmacologic Treatments Pharmacologic Treatments Alternative Medicine Surgery Articular Cartilage Composition Chondrocytes Water Collagen Proteoglycans Non-Collagenous Proteins and Glycoproteins Structure Zones Regions Chondrocyte-Extracellular Matrix Interaction The Impact of Osteoarthritis on Articular Cartilage Integrin α1β1 and Osteoarthritis Integrin α1β1 and Spontaneous Osteoarthritis Integrin α1β1 and the Potential Mechanisms of Osteoarthritis Transforming Growth Factor β Receptor Signalling Epidermal Growth Factor Receptor and Osteoarthritis Rationale...33 CHAPTER TWO: INTEGRIN Α1Β1 PROTECTS AGAINST SIGNS OF PTOA IN THE ARTICULAR CARTILAGE OF THE KNEE THROUGH A MECHANISM THAT INVOLVES EGFR SIGNALLING Introduction Materials and Methods...38 v

6 2.2.1 Animals Surgery Erlotinib Administration Euthanization and Tissue Preparation Micro-Computed Tomography Histology Statistical Analysis Results Animals Subchondral Bone Calcified Meniscus Calcified Medial Collateral Ligament Fabella Trabecular Bone Cartilage Synovitis Discussion Funding Acknowledgements...63 CHAPTER THREE: BEHAVIOURAL TESTING Introduction Materials and Methods Statistical Analysis Results Discussion...69 CHAPTER FOUR: DISCUSSION Hypothesis and Specific Aims Summary of Key Findings Integrin α1β1 Offers Less Protection Against PTOA Than Spontaneous OA Dampening Epidermal Growth Factor Receptor Signalling Protects Against PTOA in a Sex dependent Manner Female Mice are Protected from Post-traumatic Osteoarthritis up to 8 Weeks Post-surgery Strengths and Limitations Animal Model Erlotinib Administration Skeletal Maturity Incorporation of Behavioural Tests Future Studies Conclusion...82 REFERENCES...84 APPENDIX A: DMM EXPERIMENT TIMELINE vi

7 APPENDIX B: TARCEVA PREPARATION PROTOCOL APPENDIX C: MICE SACRIFICE PROTOCOL APPENDIX D: HISTOLOGY AND MICROCT PROTOCOL APPENDIX E: MICROCT ANALYSIS APPENDIX F: MICROCT THICKNESS ANALYSIS vii

8 List of Tables Table 2.1. The frequency and severity of calcified medial collateral ligaments (cmcl) in murine knees as a function of time post-surgery, sex, genotype, surgery to destabilise the medial meniscus (DMM), and erlotinib treatment. Calcification was scored for bone volume and density, and summed for a maximum calcification score of 6. Note that approximately twice as many cmcl were observed in females compared to males and in DMM compared to sham or contralateral joints. Significant differences: (p=0.009); (p=0.049); (p=0.006) Table 2.2. The frequency and severity of synovitis in murine knees as a function of time, sex, genotype, surgery to destabilise the medial meniscus (DMM), and erlotinib treatment. Synovitis was scored at all four regions (medial and lateral tibial plateau and femoral condyle) of the joint and summed. The maximum score per joint on this scale is 12. Note the four fold increase in the frequency of synovitis in the DMM compared to sham and contralateral joints, and the increased frequency of synovitis in α1-null compared to the wildtype mice. Significant differences: Table D.1 Table outlining the type of chemicals and time spent in each container of automated tissue processor Table D.2 Table outlining the type of chemicals and time spent in each container of the automated tissue staining machine viii

9 List of Figures and Illustrations Figure 1.1 (Reprinted from [3] with permission) Knee OA prevalence rates, age group and sex, broad regions, A regions = developed countries in North America, Western Europe, Japan, Australia, and New Zealand. AF = Countries in sub-saharan Africa. AM BD = developing countries in the Americas. EM = countries in the Eastern Mediterranean and North African regions. EU BC = developing countries in Europe. SEA = countries in South-east Asia. WP B = countries in the Western Pacific region Figure 1.2 (Reprinted from [111] with permission). Extracellular matrix of cartilage. Three classes of proteins exist in articular cartilage: collagens (mostly type II collagen); proteoglycans (primarily aggrecan); and other noncollagenous proteins (including link protein, fibronectin, cartilage oligomeric matrix protein) and the smaller proteoglycans (biglycan, decorin and fibromodulin). The interaction between highly negatively charged cartilage proteoglycans and type II collagen fibrils is responsible for the compressive and tensile strength of the tissue, which resists load in vivo. Abbreviation: COMP, cartilage oligomeric matrix protein Figure 1.3 (Reprinted from [120] with permission). Articular cartilage histology. (a) Van Gieson staining for collagen demonstrating superficial (S1), middle (M1) deep (D1) and calcified (C1) layers. (b) Haematoxylin and eosin staining illustrating the radial alignment and matrix organisation in the deep layers. (c) Detail showing differentiation of the pericellular microenvironment (Pm), territorial matrix (Tm) and the interterritorial matrix (Im). Bars: (a) 100 µm, (b) 50 µm, (c) 10 µm Figure 1.4 Typical confocal image of an ex vivo wild-type murine femor showing circularshaped chondrocytes in the transverse plane Figure 1.5 Spontaneous OA in integrin α1-null (α1ko) mice is apparent in the soft tissues of the knee at a younger age than in wild-type (WT) controls. (A) 40x (large) and 200x (small) images of a sagittal plane in the centre of the medial condyle of a typical 6 month (6M) and 12 month (12M) old WT and α1ko mouse knee. Sections stained with hematoxylin (nuclei black), fast green (collagen blue) and safranin-o (proteoglycans - pink). Scale bar 40x = 500 µm, 200x = 100 µm. (B) Graph of local histological score (maximum = 30) as a function of age and genotype. Each data point represents the weighted mean of three independent samples. Error bars represent 95% confidence interval, p<0.05. # = significantly different from all data points Figure 1.6 Spontaneous OA in the knees of integrin α1-null (α1ko) mice involves subchondral bone thickening and osteophyte development. (A) µct images of 6 month (6M) or 12 month (12M), wild-type (WT) or α1ko murine knees. In frontal images trabecular bone is shaded pink, subchondral bone blue and ossified menisci green. Extensive ossification is indicated in the patellar tendon (arrows) and menisci (arrow heads) in α1ko compared to WT mice. Sagittal images are from the centre of the medial condyle, with a thickness map representing thicker regions with hot colors. (B,C,D,E) Graphs of subchondral bone density (B), trabecular apparent bone density ix

10 (C), sesamoid total volume (D) and ossified meniscal volume (E) as a function of age and genotype. Each data point represents the weighted mean of three independent samples. HA = hydroxyapatite. Error bars represent 95% confidence intervals, p<0.05. # = significantly different from corresponding WT * = significantly different from 10 month of same genotype Figure 2.1 Representative microct images of left female and male murine knees at 2 and 12 weeks post-surgery (PS) that underwent either destabilisation of the medial meniscus (DMM) or sham surgery. Mice in the 12 weeks PS group were administered erlotinib (12E) or vehicle (12) daily. A-C, E-G, I-K, M-O Cortical bone at the femoral condyles and tibial plateaus is rendered transparent to show the analyzed trabecular regions. Trabecular bone is shaded blue, subchondral bone pink, and ossified menisci green. D, H, L, P Frontal cross-section taken in the middle of femoral condyle/tibial plateau contact region shaded with a thickness map with thicker regions shaded with hot colors located further right of the spectrum scale. Note the medial displacement of medial meniscus in DMM joints (A-C, I-K), larger medial meniscus and calcified medial collateral ligament at 12 weeks PS in DMM knees (B, J) with this effect lessened with erlotinib treatment in female (C) but worsened in male mice (K), and thicker subchondral bone in DMM (D, L) compared to sham (H, P) joints Figure 2.2 Representative frontal histology images of male and female α1-null and wild type (WT) left knees at 2, 4, 8 or 12 weeks post-surgery (PS). Knees underwent either destabilisation of the medial meniscus (DMM) or sham surgery and mice in the 12 weeks PS group were administered erlotinib (12E) or vehicle (12) daily. Sections were stained with hematoxylin, fast green, and safranin-o. Scale bar represents 500µm. Note the thinner and more fibrillated cartilage in the medial compartment (arrows) beginning at 4 weeks PS in male α1-null and WT DMM knees with this effect delayed until 8 weeks PS in female α1-null mice and 12 weeks PS in female WT mice. Erlotinib treatment alleviated cartilage damage in both male and female α1-null but not WT mice.. 44 Figure 2.3 Mouse mass at surgery (A) and change in mouse mass from surgery to sacrifice (B) as a function of time post-surgery (PS, weeks), sex and genotype. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 9) ± 95% confidence interval. a Different from 8, 12, and 12E weeks PS equivalent (P < 0.05). b Different from 12E weeks PS equivalent (P < 0.05). c Different from 4 and 8 weeks PS equivalent (P < 0.05). d Different from all points equivalent (P < 0.05). e Different from 2, 12, and 12E weeks PS equivalent (P < 0.05) Figure 2.4 Subchondral bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of (A, B) sex and bone or (C, D) sex and compartment. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean n 9 ± 95% confidence interval. a Different from 2, 4, and 12E weeks post-surgery (PS) equivalent (P < 0.01). b Different from all time points PS equivalent (P < 0.05). c Different from 2 and 12E weeks PS equivalent (P < 0.05). d Different from 2 and 4 weeks PS equivalent (P < 0.01). e Different from 12 weeks PS (P < 0.05). f Different from 2, 4, and 8 weeks PS (P x

11 < 0.05). g Different from 4, 8, and 12E weeks PS equivalent (P < 0.05). h Different from 8, and 12E weeks PS equivalent (P < 0.05). * Different from time-matched counterpart(s) (P <0.05). Note increased thickness with DMM in both femur (A) and tibia (B) but only in the medial compartment (C), with this diminished in erlotinib treated females but not males (A-C) Figure 2.5 Subchondral bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of compartment and bone. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (A, B n 19, C n 99, D n 39) ± 95% confidence interval. a Different from 2 and 4 weeks PS equivalent (P < 0.01). b Different from 2, 4, and 12E weeks post-surgery (PS) equivalent (P < 0.01). c Different from 2 and 12E weeks PS equivalent (P < 0.05). d Different from 4, 8, and 12E weeks PS equivalent (P < 0.05). e Different from 8, and 12E weeks PS equivalent (P < 0.05). f Different from all points (P < 0.05). g Different from contralateral equivalent (P < 0.05). * Different from surgery- or time-matched counterpart(s) (P <0.05). Note (A) increased density with DMM in the medial compartment of the femur reduced with erlotinib treatment, (C) increased bone volume with DMM in both femur and tibia but only in the medial compartment, and (D) a contrasting effect of erlotinib in the medial and lateral compartments of the tibia but not the femur Figure 2.6 Meniscal bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of (A, B) site and sex or (C) compartment. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (A, B n 9, C n 19), ± 95% confidence interval. a Different from all time points equivalent (P < 0.05). b Different from 2 and 4 weeks PS equivalent (P < 0.01). * Different from time-matched counterpart(s) (P <0.05). Note (A, B) the increase in meniscal bone volume with DMM in both anterior and posterior sites but (C) only in the medial compartment and (A, B) the reduction of this with erlotinib treatment in females but not males Figure 2.7 Fabella bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of (A-D) compartment and (C, D) sex. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 19) ± 95% confidence interval. a Different from 2 and 4 weeks PS equivalent (P < 0.05). b Different from 12E weeks PS equivalent (P < 0.05). c Different from 2 and 8 weeks PS equivalent (P < 0.05). d Different from 8 weeks PS equivalent (P < 0.05). e Different from 2 weeks PS equivalent (P < 0.05). f Different from 12 weeks PS equivalent (P < 0.05). g Different from 12 and 12E weeks PS equivalent (P < 0.05). * Different from time-matched counterpart(s) (P <0.05). Note (A, B) the significant effect of DMM in the medial but not lateral compartment and the reduction of this with erlotinib treatment and (C, D) the significant effect of erlotinib in females but not males, independent of surgery xi

12 Figure 2.8 Trabecular bone remodelling in murine knees 2, 4, 8, and 12 weeks post-surgery (PS) as a function of sex, compartment and bone. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 19) ± 95% confidence interval. a Different from 2 weeks PS equivalent (P < 0.05). b Different from all other points (P < 0.01). c Different from 8 weeks PS equivalent (P < 0.05). d Different from all other points except 8 weeks PS equivalent (P < 0.01). e Different from 2 and 12E weeks PS equivalent (P < 0.05). * Different from timematched counterpart(s) (P <0.05). Note the significant effect of erlotinib in females but not males on all parameters and a delayed response in males compared to females in some femoral parameters Figure 2.9 Trabecular bone remodelling in murine knees 2, 4, 8, and 12 weeks post-surgery (PS) as a function of genotype, sex and bone. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 9) ± 95% confidence interval. a Different from 2 and 12E weeks PS equivalent (P < 0.05). b Different from 4 and 8 weeks PS equivalent (P < 0.05). c Different from 8 weeks PS equivalent (P < 0.05). d Different from 12, and 12E weeks PS equivalent (P < 0.05). e Different from 2, 4, and 12E weeks PS equivalent (P < 0.05). f Different from 8 and 12 weeks PS equivalent (P < 0.01). * Different from time-matched counterpart(s) (P <0.05). Note (A, B) the significant effect of erlotinib in females but not males and (B, D) genotypic differences in tibial parameters in females at early time points Figure 2.10 Maximum histology osteoarthritis score (out of 6) of murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of sex, genotype, and region. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. MFC Medial Femoral Condyle. MTP Medial tibial plateau. LFC Lateral femoral condyle. LTP Lateral Tibial Plateau. Data points represent mean (n 4) ± 95% confidence interval. a Different from 2 and 12 weeks PS (P < 0.01). b Different from all points of equivalent region (P < 0.05). c Different from 2, 8, and 12 weeks PS equivalent (P < 0.05). d Different from 2 weeks PS equivalent (P < 0.01). e Different from 2 and 4 weeks PS (P < 0.01). f Different from 2, 4, and 8 weeks PS equivalent (P < 0.01). * Different from time-matched medial compartment (P <0.05). Note the significant effect of DMM in the medial compartment, the erlotinib effect in αl-null but not wildtype mice, and a delayed response in females compared to males and in WT females compared to α1-null females Figure 2.11 Summed histology osteoarthritis score (out of 48) of murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of sex, genotype, and region. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. MFC Medial Femoral Condyle. MTP Medial tibial plateau. LFC Lateral femoral condyle. LTP Lateral Tibial Plateau. Data points represent mean (n 4) ± 95% confidence interval. a Different from 2 and 4 weeks PS equivalent (P < 0.05). b Different from 2, 12, and 12E weeks PS equivalent (P < 0.01). c Different from 2, 4, and 12E weeks PS equivalent (P < 0.05). d Different from all points of equivalent region except 12E (P < 0.01). e Different from all points of equivalent region (P < 0.05). * Different from time-matched medial compartment (P <0.05). Note the significant effect xii

13 of DMM in the medial compartment, the erlotinib effect in αl-null but not wildtype mice, and a delayed response in females compared to males and in WT females compared to α1-null females Figure 3.1 Time spent immobile (A) and climbing (B) as a function of treatment (vehicle or erlotinib) over 23 hours of test duration. Data points represent mean of n = 3 trials for N = 12 mice ± 95% confidence interval. Note the trend (though statistically insignificant) of less time spent immobile (A) and more time spent climbing (B) in erlotinib compared to vehicle treated mice Figure 3.2 Time spent in locomotion at 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of treatment (erlotinib or vehicle) during their most active time (8pm 12am). Data points represent mean (N = 6 mice) ± 95% confidence interval. Note the trend (though statistically insignificant) of less time spent in locomotion for vehicle treated joints at 8 and 12 weeks PS compared to 4 weeks PS Figure 3.3 Conservative sample size determination for repeated measures analysis Figure C.1 After tenting and making the incision the skin is pulled off Figure C.2 The limb has to lie flat on the table before it can be removed Figure C.3 Limb is removed above the knee as close to the hip as possible Figure C.4 Removal of the foot at the ankle Figure C.5 Sample ready to be put in the freezer Figure C.6 Folding the tail between the paws of the mouse Figure C.7 Mouse wrapped in first piece of gauze (vertical fibers) Figure C.8 Mouse wrapped with second piece of gauze (horizontal fibers) Figure C.9 Mouse wrapped in gauze and placed in Ziploc bag with air removed Figure D.1 Descriptive diagram and photos of the foam used. The numbers in top view represent the positions of each joint Figure E.1 Micro-computed tomography image showing the four positions of joints in a single scan Figure E.2 Micro-computed tomography images showing the slice with largest anterior or posterior menisci Figure E.3 The contour button Figure E.4 The contour tool xiii

14 Figure E.5 Micro-computed tomography image showing the meniscus contour Figure E.6 Micro-computed tomography image showing the corrected contour Figure E.7 The editing tool Figure E.8 Micro-computed tomography images showing growth plates appearing as well as a slice to begin the contour Figure E.9 Micro-computed tomography image showing the contour of femoral trabeculae Figure E.10 Micro-computed tomography image showing where the last femoral trabeculae appear Figure E.11 Micro-computed tomography image showing where the first tibial trabeculae appear Figure E.12 Micro-computed tomography images showing the contouring of tibial trabeculae Figure E.13 Micro-computed tomography images showing how to modify the tibial trabeculae Figure E.14 Micro-computed tomography images showing how to contour near the growth plate (green). The growth plate appearing is marked in red Figure E.15 Micro-computed tomography images showing where growth plates appear and where to contour Figure E.16 Micro-computed tomography image showing how to contour the trabeculae box to separate the medial and lateral compartment Figure E.17 Micro-computed tomography image showing how to contour the trabeculae box to separate the medial and lateral compartment Figure E.18 Micro-computed tomography image showing the contouring of the first slice containing the tibial subchondral bone Figure E.19 Micro-computed tomography image showing the contouring of the 12 th slice containing the tibial subchondral bone Figure E.20 Micro-computed tomography image showing the contouring of the 1 st and 12 th slice containing the respective subchondral bone Figure E.21 The object properties window Figure F.1 Micro-computed tomography model showing the measurement of a medial femoral condyle xiv

15 Figure F.2 Micro-computed tomography model showing the measurement of the 25% of the measured length of the femoral condyle Figure F.3 Micro-computed tomography model showing the measurement of the thickness at the 25% of the measured length of the femoral condyle xv

16 List of Symbols, Abbreviations and Nomenclature Symbol Definition OA Osteoarthritis PTOA Post-traumatic osteoarthritis NSAIDs Nonsteroidal anti-inflammatory drugs PCM Pericellular matrix WT Wildtype TRPV4 Transient receptor potential vanilloid 4 IL-1 Interleukin-1 GAG Glycosaminoglycan PRG4 Proteoglycan 4 ACLT Anterior cruciate ligament transection EGFR Epidermal Growth Factor Receptor MSCs Mesenchymal stem cells α1ko Integrin α1-null TCPTP T cell protein tyrosine phosphatase TGF-α Transforming growth factor α Mig6 Mitogen-inducible gene 6 DMM Destabilisation of medial meniscus LABORAS Laboratory Animal Behaviour Observation Registration and Analysis System PS Post-Surgery α Alpha β Beta TGF-β Transforming growth factor beta TβR Transforming growth factor beta receptor MicroCT Micro-computed tomography SMI Structure model index cmcl Calcified medial collateral ligament Y845 Tyrosine 845 ROS Reactive oxygen species xvi

17 Epigraph Success is not final, failure is not fatal: it is the courage to continue that counts. Winston Churchill xvii

18 Chapter One: Literature Review 1.1 Osteoarthritis Osteoarthritis (OA) is the most common type of degenerative joint disease and is characterized by the gradual loss of articular cartilage and alteration of its surrounding tissue [1, 2]. It affects a wide range of joints, including the hand, knee, hip, and spine, with a greater debilitating effect in weight bearing joints [1, 2]. OA can be classified as primary or secondary. Primary OA refers to idiopathic OA where no obvious cause can be attributed to the disease, whereas secondary OA involve a distinct cause (i.e. joint trauma) that likely initiated the OA [1, 2]. Despite there being no specific cause, risk factors for primary OA include such as age, genetic predisposition, and obesity Epidemiology Osteoarthritis is regarded as the leading cause of disability and economic burden worldwide, especially in the developing countries of the world (Figure 1.1) [3, 4]. The World Health Organization estimates that approximately 9.6% of male and 18% of females 60 years and over are suffering from symptomatic OA worldwide [5]. Due to the increase in population and their life expectancy, the incidence of OA is projected to rise over time. In 2011, more than 4.4 million people suffered from OA and Canada spent 10 billion dollars in direct costs alone [6]. If no improvements are seen in the prevention and/or care of OA, the prevalence is estimated to more than double to 10.4 million people by 2040 [6]. 1

19 Figure 1.1 (Reprinted from [3] with permission) Knee OA prevalence rates, age group and sex, broad regions, A regions = developed countries in North America, Western Europe, Japan, Australia, and New Zealand. AF = Countries in sub-saharan Africa. AM BD = developing countries in the Americas. EM = countries in the Eastern Mediterranean and North African regions. EU BC = developing countries in Europe. SEA = countries in South-east Asia. WP B = countries in the Western Pacific region. Post-traumatic OA (PTOA), classified as secondary OA, occurs when an individual develops OA following a serious injury to a joint. It accounts for approximately 12% of all incidences of OA, and this proportion is expected to increase given the rise in participation and injury prevalence in youth sports [7-9]. In just 12 years ( ), there was a 400% increase in pediatric knee injuries, and approximately 50% of these individuals developed OA associated pain within 10 to 20 years of injury [9, 10]. When taken together, the average age of onset of OA is estimated to decrease over time. The clearly defined initiation of OA at the time of injury enables these high risk individuals to be identified and may allow for early intervention before 2

20 any signs of OA arise. Despite this treatment window, there are still no effective strategies to delay or slow the progression of PTOA Signs and Symptoms The term osteoarthritis comes from the Greek words osteon for bone, arthron for joint, and itis for inflammation [1]. Surprisingly, however, the most striking macroscopic change observed is not the bone, but the articular cartilage of the joint [1]. In severe OA, full thickness cartilage loss is found, together with osteophyte formation, subchondral bone sclerosis, joint space narrowing, and in some cases synovitis [1, 2, 11]. As the disease progresses, such damage to the joint may induce a wide variety of symptoms such as pain, joint stiffness, swelling, and limited range of motion [1, 2]. Interestingly, however, the reported relationship between the presence of radiographic OA and the symptoms is not consistent. The severity of radiographic OA does not necessarily predict the level of experienced symptoms, or vice versa, and sometimes an individual with mild radiographic OA reports a higher degree of pain than another individual with severe radiographic OA [12-14]. Furthermore, due to the lack of symptoms in its initial stages, the detection of OA often occurs years after joint damage has begun. Together, these problems hamper the early diagnosis and treatment of OA Risk Factors The etiology of OA is complex and is not clearly defined. Although there may be a distinct contributing factor in the case of secondary OA, in the majority of cases, OA is thought to be a result of an interaction between systemic and local risk factors related to OA [2, 15, 16]. For example, an individual at a higher risk of OA from systemic risk factors (i.e. elderly) may not 3

21 develop the disease until a local risk factor is present, such as joint injury. Due to the unique functioning environment of each joint (i.e. level of loading), the systemic and local risk factors may have varying influences on the disease depending on its location Systemic Age, sex, and ethnicity/race are some of the strongest risk factors associated with the onset of OA. It is well documented that the incidence and severity of OA is significantly increased with age, especially past 30 years of age in the case of knee OA (Figure 1.1) [3, 17-20]. Zhang and Jordon (2010) predicted that this may be due to the biological changes, such as weak muscle strength and oxidative changes, that make the tissue vulnerable to damage and hinder its ability to repair [15]. Sex is an interesting factor to consider as its effect on the risk of OA is variable from site to site. The incidence of knee and hip OA in males is significantly less than that of females, but not in hand OA [21]. Similarly, females are found to have more severe knee OA than males, but no sex difference in severity was found in hip and hand OA [21]. Due to how the timing of menopause coincides with the increase in prevalence of OA in females, some hypothesized that hormones influence the progression of OA. Several studies indicate an increase in joint laxity during menstrual cycle in young, healthy individuals, thus providing support for hormonal influence on OA [22-24]. However, contradicting and therefore inconclusive results are reported in the literature regarding the risk of OA in menopausal women undergoing estrogen therapy [25-29]. Further studies are needed to clarify the direct relationship between sex hormones and its effect on OA. 4

22 Numerous reports suggest that both the prevalence and the severity of OA vary among different racial/ethnic groups. However, a single race or ethnicity may not have an overall lower risk of developing OA. For instance, the Beijing OA Study revealed that hip and hand OA occurred less frequently in Chinese compared to the Caucasians in the Framingham Study, but a higher prevalence of knee OA was found among Chinese women compared to the Caucasian women (no difference among males) [30-32]. In the Johnston County OA Project, African Americans were found to have more severe knee OA than their Caucasian counterparts [33]. Interestingly, once adjusted for demographic and clinical factors, this difference was strengthened in males but weakened in females [33]. In contrast, a study performed in Russia showed that there is no difference in the prevalence and severity of hand OA between two unique ethnic groups, Russians and Buryats, residing in the same geographical area [34]. This is especially fascinating when the same authors previous research result is considered, where they found a significant difference in the prevalence and severity of hand OA among a single ethnic group living in five different communities [35]. The varying trend of OA prevalence among different ethnic or racial groups implies that the susceptibility of ethnic or racial groups toward OA may involve other factors such as sex and the environment Local The strongest local risk factors associated with OA development include joint overload from obesity, trauma from injury or surgery, and repetitive use from certain occupations. Obesity has been identified as a strong risk factor of knee OA ever since the 1980 s [36]. Blagojevic et al. (2010) showed in a meta-analysis that obese or overweight individuals have almost three times higher risk of developing knee OA than normal weight individuals [37]. In addition to the 5

23 absolute weight of an individual, the change in weight has also been associated with the incidence of OA. In the Framingham Study, a weight-loss of 5 kg in overweight and obese women reduced the risk of developing symptomatic knee OA by 41 percent, and weight gain, in contrast, increased the risk of OA, but only by 20 percent [38]. Interestingly, the risk of OA for women with body mass index of less than 25 was not affected by either weight-loss or gain [38]. The impact of obesity on the risk of developing OA in other joints is not as strong or inconclusive. There is a significant amount of support for the association between knee injury and the onset of knee OA. A recent meta-analysis revealed that all but one of 13 cohort studies showed an increased risk of developing knee OA with previous injury [39]. Knee injuries leading to OA vary significantly in type and severity. For instance, intra-articular fractures pose a significant trauma to the joints involved and may cause permanent changes in stability and alignment of the joint that can further damage the tissues [40-43]. The risk of developing PTOA following an intra-articular fracture at the tibia is shown to be as high as 75 percent [41]. Other common contributing injuries include meniscal lesion and anterior cruciate ligament tear, which are often surgically treated [44, 45]. A strikingly higher proportion (82%) of individuals with radiographic knee OA were found to have meniscal damage compared to those without OA (25%), and the prevalence of meniscal damage increased with the severity of OA [46]. Repetitive joint use at work is another strong risk factor for OA. Occupational work accounts for a large proportion of an individual s daily routine, and the type of work is associated with the specific site of OA. Occupations requiring frequent lifting, kneeling or squatting, such as farmers and construction workers, were reported to have a higher prevalence of hip and knee OA [47, 48]. An interesting study of the prevalence of hand OA among textile workers showed that 6

24 not only were the impairments concentrated on the hand involved in the task, but the workers with a task requiring finer digit movement had more OA at distal interphalangeal joints than that of workers whose task required a power grip [49]. Unfortunately, an effective preventative measure against OA is not yet in place to help those at risk Treatments The contemporary measures to manage the progression of OA lack a permanent cure, and are mainly focused on alleviation of pain and improvement of function. In general, the treatment options fall into four categories: nonpharmacologic, pharmacologic, alternative medicine, and surgery [50]. Although the surgical approach is clinically shown to yield the best results, due to the limited lifespan of prostheses, more conventional and less-invasive nonpharmacologic and pharmacologic treatments should be sought first Nonpharmacologic Treatments Those with mild symptoms of OA are typically advised to begin with nonpharmacologic therapy, such as exercise, aimed at improving muscle strength and range of motion [50]. Two randomized controlled trials showed a significant improvement in short-term outcomes with monitored exercise compared to a group with no exercise [51, 52]. The exercise programs in both studies consisted of muscle strengthening and range of motion exercises, using tools such as graded elastic bands, which lasted approximately 30 minutes per session [51, 52]. The number of sessions per week ranged from one to three sessions per week depending on the pain level [51, 52]. This benefit was observed as early as six months from the exercise start date and continued until the effect was diminished at 36 months [51, 52]. Both land-based and water-based exercises 7

25 show reduction of pain and improvement of function, with greater benefits with the land-based exercises [53, 54]. Other nonpharmacologic treatments include weight-loss and bracing, and are both considered beneficial to a certain degree [38, 50] Pharmacologic Treatments Pharmacologic treatments are primarily used for pain reduction and are often prescribed alongside with other treatment options. In general, an initial trial of acetaminophen is recommended for mild to moderate OA patients due to its affordability, efficacy, and low toxicity [55, 56]. Numerous studies have indicated that although acetaminophen is effective against OA pain, it is not as effective as other alternatives in alleviating severe OA pain [57-59]. One alternative is the family of nonsteroidal anti-inflammatory drugs (NSAIDs), which has been shown to be significantly more effective than acetaminophen in treating severe OA, albeit more costly and having more side-effects [57-59]. If the patient does not respond to either acetaminophen or NSAIDs, opioids may be prescribed by a physician. Due to the narcotic properties of opioids, it is not prescribed unless other alternatives of alleviating pain are found to be effective [50]. However, oral treatments are sometimes limited in its use in geriatric population or in individuals with end-organ damage due to adverse effects [60, 61]. Topical analgesic is an alternative to oral agents that offer several advantages including site-specific drug delivery and reduced systemic side effects [62]. There are several topical NSAIDs that have been tested in the treatment of chronic OA pain, such as diclofenac and ketoprofen [63, 64]. Topical application of diclofenac has been shown to be superior to placebo and as effective as some oral NSAIDs treatments (i.e. ibuprofen) in alleviating knee OA, with lower potential for systemic complications [63]. Similarly, 6 week-treatment of ketoprofen gel 8

26 was also shown to be superior to placbo and as effective as celecoxib in knee OA pain reduction [64]. Given the efficacy and safety of topical NSAIDs in treating OA pain, the American College of Rheumatology recommends the use of topical NSAIDs for initial management of pain in hand and knee OA [65]. An interesting substitute to oral and topical drugs is an intra-articular injection of corticosteroids or hyaluronic acid. Corticosteroids are presumed to reduce inflammation, whereas hyaluronic acid is thought to provide elastoviscous properties to the synovial fluid [66]. Metaanalyses of the efficacy of both injections suggested that corticosteroids provide relief that can last from one to two months while hyaluronic acid provide relief for a much longer period of time, from one to six months [67-69]. Nevertheless, both injection procedures are merely a temporary solution to the progressively degenerative disease Alternative Medicine Alternative medicine may involve a variety of practices, including acupuncture and health supplements. There is a growing interest of testing the efficacy of acupuncture in pain management, partly due to the safety and rare adverse effects of its use [70-73]. In 2007, a metaanalysis of the use of acupuncture in treating knee OA showed minimal or no clinically relevant effects compared to the sham group [74]. In a more recent review performed by Corbett et al. (2013), the authors suggest that acupuncture can be considered effective in alleviating knee OA pain for a short period of time, up to 8 weeks [75]. However, the same authors also stressed that many of the trials included in the network meta-analysis were only assessing short-term effects and had a high risk of bias due to the lack of blinding of patients [75]. Provided this, the benefits, if any are present, of acupuncture in knee OA pain alleviation need further clarification. 9

27 The most commonly used supplements for OA are glucosamine and chondroitin. Despite the common use, however, there is no conclusive support that glucosamine, chondroitin, or a combination of both reduces pain compared to placebo [76-78] Surgery Total joint replacement surgeries are an option for those with on-going symptoms unable to be treated by other means or those diagnosed with end-stage OA that significantly limits their function. Given the high invasiveness of the surgery, the post-operative results are quite striking. After a hip or knee arthroplasty, the patients gain a significantly improved mobility and a quality of life that is equal to, if not exceeding, the population norm [79]. However, as mentioned previously, arthroplasty should be the last resort in treating OA primarily because of the short durability of prostheses. The survival analyses of prostheses showed that the hip devices function well for 15 to 20 years and the knee devices last for approximately 15 years [80, 81]. Revision replacement surgeries can be performed; however, the beneficial outcomes of a revision surgery are not as marked as that of the primary arthroplasty [82]. Furthermore, the revision surgery is found to be associated with higher rates of mortality, hip dislocation, and infection [82]. Other surgical interventions are also available, such as arthroscopic debridement, but either with much less success than arthroplasty or without evidence to show any advantage over non-invasive treatments [83]. Perhaps more importantly, all of the above mentioned treatment options only provide a temporary solution to alleviate pain and/or improve joint function [84]. An effective treatment for OA addressing the signs and progression of the disease at the tissue and cellular level is 10

28 required, perhaps by direct pharmaceutical targeting of specific molecules which promote homeostasis of joint health. 1.2 Articular Cartilage Synovial joints are complex structures enveloped in a joint capsule and involve the interaction of multiple tissues including articular cartilage, subchondral bone, meniscus, and ligaments. The articulating surface of all human synovial joints is covered by a thin layer of articular cartilage that is at most a few milimeters thick. Despite being thin, the cartilage offers a great deal of mechanical advantage to the joint, including reduction of friction and distribution of load [85]. Unlike human-made machinery, articular cartilage has an outstanding durability, providing normal joint function up to 80 years or longer in many individuals. Up to a certain degree, it is able to detect and respond to changes in use; however, due to the lack of vasculature, nerves, and the lymphatic system, articular cartilage is severely limited in its ability to heal [85]. With limited, if any, access to oxygen, the cartilage is shown to be primarily reliant on anaerobic metabolism, and thus have a low level of metabolic activity compared to other vascular tissues such as muscle or bone [85, 86]. At a glance, the articular cartilage may perhaps appear to be a simple inert tissue, but detailed studies suggest otherwise. It has a well-defined structure and makes use of complex interactions between chondrocytes and their matrix to maintain the integrity of the tissue [85] Composition Despite the variance in thickness, matrix composition, and mechanical properties of articular cartilage in the same joint, among joints, and across species, all articular cartilage is 11

29 similar in its components, structure, and function [87]. The articular cartilage is composed of highly specialized cells called chondrocytes, matrix fluid, and a matrix macromolecular framework [88]. Only about one percent of articular cartilage in humans is occupied by chondrocytes, but the cell density is greater in other species, such as mouse, whose cartilage is much thinner [89, 90]. Approximately percent of the articular cartilage is water, making it one of the most hydrated tissues in our body [88]. Among the percent dry weight of the tissue, collagen contributes 60 percent, proteoglycans about percent, with non-collagenous proteins and glycoproteins taking up the rest [85] Chondrocytes All chondrocytes possess endoplasmic reticulum and golgi membranes required for matrix synthesis [85]. Chondrocytes attach themselves to the self-produced matrix, allowing them to be receptive to mechanical, electrical, and physiochemical changes in its environment [85]. The activity and function of chondrocytes is dependent on the skeletal maturity of an individual. Prior to skeletal maturity, the chondrocytes divide and synthesize matrix to enlarge the articular surface [91]. In contrast, after skeletal maturity, chondrocytes rarely divide or expand the volume of the tissue [92]. Rather, they continue to remodel the macromolecular framework by degrading and replacing fragmented molecules, thus providing an on-going maintenance of the articular cartilage until chondrocyte senescence [91]. This delicate balance of degradation and synthesis of matrix macromolecules can be altered by joint immobilisation, injury, and aging, which may limit the chondrocytes ability to maintain the integrity of the tissue [93-95]. Interestingly, a matrix comprised of appropriate concentrations of water and macromolecules but lacking in chondrocytes does not replicate the properties of articular cartilage [85]. This further suggests 12

30 that chondrocytes play an important role in synthesis and assembly of macromolecules, and allows the tissue to adjust its properties in response to use of the joint Water As the most abundant component of articular cartilage, water substantially influences the mechanical properties of articular cartilage. The proteoglycans trapped within the collagen framework of the tissue imbibe water, and thus provide the tissue with its water retention properties [85, 96-98]. The loading of the joint causes some of the water in the matrix to move out of the articular cartilage, but it is drawn back into the tissue upon subsequent unloading of the joint. This capacity of the tissue to retain water gives it the ability to bear loads of significant magnitude [96]. In addition, the water flow also provides lubrication of the tissue at the articular surface as well as transportation and distribution of nutrients from the synovial fluid to chondrocytes [96] Collagen The majority of the matrix macromolecular framework consists of collagen, and multiple collagen types are present in articular cartilage, specifically types II, VI, IX, X, and XI [85, 99]. Among them, collagen type II is the primary collagen found in articular cartilage, accounting for approximately 90 to 95 percent [85]. The fibrous mesh formed by collagen types II, IX, and XI provide the tissue with tensile strength and entraps the proteoglycans, giving it water retention properties [85]. The function of type X collagen remains uncertain, but its primary presence near the calcified cartilage zone and the hypertrophic zone of the growth plate suggest its relevance to cartilage calcification [85]. Lastly, type VI collagen is concentrated in the areas immediately 13

31 surrounding the chondrocytes referred to as the pericellular matrix (PCM), suggesting its importance in cell-matrix interaction [100, 101]. Mechanical testing of the PCM of femoral head articular cartilage showed that the stiffness of PCM is significantly reduced in mice lacking in type VI collagen compared to the wild-type (WT) mice [102, 103]. Zelenski et al. (2014) also showed that the loss of collagen VI led to alterations in transient receptor potential vanilloid 4 (TRPV4) mediated chondrocyte mechano-osmotic signalling, thus highlighting the important role of collagen VI in normal mechano-transduction between chondrocytes and PCM [103]. Furthermore, mutations in type VI collagen genes in humans have been linked to muscle disorders such as Bethlem myopathy and Ullrich muscular dystrophy, which compromises the joint integrity by increasing joint laxity [104, 105]. In addition to the maintenance of PCM integrity, collagen VI also plays a role in remodelling of the articular cartilage. During the early stages of OA, chondrocytes exposed to interleukin-1 (IL-1) have increased sequestration and retention of collagen VI within the PCM [106]. Also, Smeriglio et al. (2015) observed that direct application of soluble collagen VI significantly increased the proliferation rate of both normal and OA chondrocytes (or chondrogenic progenitor cells) in vitro [107]. These alterations in the mechanical properties of the joint as well as the potentially decreased cell proliferation in collagen VI null mice are hypothesized to contribute to the accelerated development of osteoarthritic changes in both bony and soft tissue parameters of the hip joint [102, 107]. In contrast, another study of collagen VI null mice reports that these mice develop delayed and less severe cartilage degeneration but early and severe osteophytes at the knee [108]. The clinical evidence of Bethlem myopathy in humans show a disparity in the musculoskeletal alterations at different sites, including proximal muscle weakness and distal joint contractures, which is consistent with the different consequences of collagen VI deficiency at different joints 14

32 [105]. These results suggest that collagen VI is an important factor for normal development of the musculoskeletal system, but may have different roles at different sites in the body Proteoglycans Proteoglycans have a protein core to which one or more glycosaminoglycan (GAG) chains are attached, and its functions may vary depending on the type of GAG chains attached [109]. The majority of the proteoglycans present in the cartilage matrix are aggrecans, accounting for 90 percent of cartilage proteoglycan mass (Figure 1.2) [85]. Aggrecans have a large number of chondroitin-sulfate and keratan-sulfate GAG chains attached to the protein core, and multiple aggrecans associate with a central hyaluronic acid and small non-collagenous proteins to form proteoglycan aggregates [110]. As mentioned previously, these proteoglycan aggregates are immobilized within the collagenous fibril meshwork and allow the articular cartilage to swell via the Donnan Effect [85, 97]. 15

33 Figure 1.2 (Reprinted from [111] with permission). Extracellular matrix of cartilage. Three classes of proteins exist in articular cartilage: collagens (mostly type II collagen); proteoglycans (primarily aggrecan); and other noncollagenous proteins (including link protein, fibronectin, cartilage oligomeric matrix protein) and the smaller proteoglycans (biglycan, decorin and fibromodulin). The interaction between highly negatively charged cartilage proteoglycans and type II collagen fibrils is responsible for the compressive and tensile strength of the tissue, which resists load in vivo. Abbreviation: COMP, cartilage oligomeric matrix protein. For every negative charge of GAG chain, a counter-ion must be present to maintain electroneutrality, and thus the total ion concentration of the tissue is always greater than that of the synovial fluid [97]. The osmotic pressure gradients resulting from differing ion concentrations drive the water from the synovial fluid into the tissue, and this effect is referred to as the Donnan effect [97]. The rest of the cartilage proteoglycan mass is attributed to non-aggregating proteoglycans. Rather than forming aggregates, these smaller proteins are capable of binding to other macromolecules and aid or alter their functions [85, 96]. Decorin and fibromodulin interact 16

34 with type II collagen and seem to be involved in stabilizing the collagen meshwork [109]. Biglycan, on the other hand, is localized in the PCM and thus is hypothesized to interact with type VI collagen [109] Non-Collagenous Proteins and Glycoproteins Both universal and cartilage-specific non-collagenous proteins and glycoproteins have been identified, but in general, their function is poorly understood. Cartilage oligomeric matrix protein is one example of these proteins that is suggested to only exist in articular cartilage and has the ability to attach to chondrocytes [112, 113]. Interestingly, this oligomeric protein is found at high concentrations in the synovial fluid following a knee trauma, and thus is suggested as a potential marker of cartilage degradation [114]. Other non-collagenous proteins, such as fibronectin and tenascin, are found in a variety of tissues and are thought to be involved in the maintenance of matrix integrity [85]. Lubricin, also referred to as proteoglycan 4 (PRG4), is a mucin-like glycoprotein secreted both by the synoviocytes and chondrocytes which help to lubricate the articular boundaries of a joint [115, 116]. Ludwig et al. (2012) showed that PRG4-deficient human OA synovial fluid has a significantly diminished lubricating ability and that the supplementation of PRG4 to these joints restored its lubricating function [117]. Complimenting results were found in an in vivo study where it reported a protective effect of chondrocyte-specific overexpression of lubricin against the anterior cruciate ligament transection (ACLT) model of PTOA in mice [118]. 17

35 1.2.2 Structure Despite only being two to four millimetres thick, human articular cartilage has a complex structure and organization that varies in composition and properties of the matrix depending on depth (zones) from the surface and distance from the cell (regions) Zones Articular cartilage consists of a spectrum of four different zones, which are referred to as the superficial, middle (or transitional), deep, and the calcified zones (Figure 1.3). Although the boundaries of each zone cannot be clearly defined, the distinct morphological features of each zone have been shown to significantly influence its physiological properties [119]. Figure 1.3 (Reprinted from [120] with permission). Articular cartilage histology. (a) Van Gieson staining for collagen demonstrating superficial (S1), middle (M1) deep (D1) and calcified (C1) layers. (b) Haematoxylin and eosin staining illustrating the radial alignment and matrix organisation in the deep layers. (c) Detail showing differentiation of the pericellular microenvironment (Pm), territorial matrix (Tm) and the interterritorial matrix (Im). Bars: (a) 100 µm, (b) 50 µm, (c) 10 µm. 18

36 The superficial zone is the thinnest zone of the tissue and is in direct contact with the synovial fluid at all times. The chondrocytes in this zone are elliptical in shape (in longitudinal view. They are circular in transverse view) and are aligned parallel to the articular surface (Figure 1.3 and Figure 1.4). Figure 1.4 Typical confocal image of an ex vivo wild-type murine femor showing circularshaped chondrocytes in the transverse plane. A relatively high concentration of collagen and low concentration of proteoglycan is found in the superficial zone, and this dense network of collagen fibrils affects the permeability of the tissue to molecules as well as the tensile strength and stiffness of the tissue [85, 121]. In a canine model of OA, the disruption and remodelling of the collagen network in the superficial zone was shown to be one of the first major events that occur in early OA [122]. When taken together, it is 19

37 postulated that, although relatively thin, the superficial zone contributes significantly to the tissue s mechanical properties, and the deterioration of this zone may leave the tissue vulnerable to irreversible damage. The transitional zone is twice or three times thicker than the superficial zone, representing approximately 40 to 60 percent of the tissue (Figure 1.3). In contrast to the elliptical shape of chondrocytes in the superficial zone, the cells in the transitional zone are spherical and less dense [96]. Perhaps due to the larger volume of matrix required to be maintained by the cells, the chondrocytes in this zone are shown to possess a higher concentration of synthetic organelles [85]. Compared to the superficial zone, the transitional zone matrix consists of larger-diameter collagen fibres, higher concentration of proteoglycan molecules, and a lower concentration of water [85]. As the last remaining layer of uncalcified articular cartilage, the deep zone is considered to provide the most support for compressive forces [96]. In support of this theory, it has been shown that the collagen fibrils of largest diameter, as well as columns of spheroidal chondrocytes, are arranged perpendicular to the articular surface [85]. Furthermore, the compressive modulus of bovine articular cartilage has been shown to be depth-dependent, where the compressive modulus is highest in the deepest layer [123]. A theoretical model developed by Wu and Herzog (2002) showed that this heterogeneous mechanical property of articular cartilage is attributed to the shape and orientation of chondrocytes and its surrounding collagen [124]. In agreement with the experimental results, Wu and Herzog showed that the Young s modulus of articular cartilage is depth-dependent where the modulus of deep zone is higher than that of the superficial zone by a factor of [124]. Interestingly, the simulation also suggests that if the long axes of soft cells are parallel to the collagen fibres, it stiffens the structure in the direction of the fibres and softens 20

38 the structure in the direction normal to the fibres [124]. This resembles the characteristics of the deep layer of articular cartilage. Together, these results provide evidence for the intimate relationship between structure and function of a tissue. At the end of the articular cartilage spectrum is the calcified cartilage zone, which serves as a transitional zone between the soft cartilage and the underlying subchondral bone. A small population of cells reside in this layer, and they are often found to be hypertrophic [96]. One of the primary functions of this zone is to anchor the collagens of the deep zone to the subchondral bone, thereby stabilizing the cartilage-bone attachment [96] Regions Based on its proximity to cells, and content and structure, the matrix within a zone can be further divided into three distinct regions: pericellular, territorial, and inter-territorial regions (Figure 1.3). As the name pericellular suggests, this region envelops the cell surface allowing direct and physical bonds to form between the matrix and the cellular membrane. A rich supply of proteoglycans, anchorin CII, and type VI collagen are found in this region which provide support to the cell, however it lacks in fibrillary collagen [85, 101, 125]. The physical interaction present here between the cell and its PCM may provide critical mechanical information to the cell during joint-loading [85]. The collagenous fibrils of the territorial region surround the PCM of a single or a column of chondrocytes, and appears to provide additional protection for the cells against mechanical stress [85]. 21

39 The rest, and indeed the majority, of the matrix are classified as the inter-territorial region. Unlike that of the territorial region, the large-diameter collagen fibrils found in this region have a zone-dependent orientation, as mentioned previously, and this provides the heterogeneous depth dependent mechanical properties of articular cartilage Chondrocyte-Extracellular Matrix Interaction The adhesion of chondrocytes to its extracellular matrix occurs primarily via receptorligand bonding such as membrane bound protein attachment to different types of collagens [126]. Anchorin CII is a glycoprotein residing in the cellular membrane that binds to collagen II and helps to anchor the chondrocytes to its matrix [125]. Mollenhauer et al. (1984) showed that the antibody against anchorin CII reduces the rate of chondrocyte attachment to collagen II, which demonstrates the important role of anchorin CII in chondrocyte-matrix interaction [125]. However, since the attachment could not be completely inhibited even with the excess of antianchorin CII, the chondrocytes must have other mechanisms of attachment to the matrix [125]. Chondrocyte-specific adhesion factor called chondronectin was also identified by Hewitt et al. in 1980 and was shown to bind to four different types (I-IV) of collagens, with the highest affinity to type II collagen [127]. Chondronectin is primarily concentrated near the cell or within the PCM and is found in both healthy and OA canine articular cartilage, suggesting that it is a part of normal chondrocyte-matrix interaction [128]. A family of transmembrane proteins called integrins also contribute to the chondrocytematrix interaction. Integrin molecules are receptors comprised of α and β subunits that bind, as a complex, to extracellular components and form a mechanical link to the cell cytoskeleton [129, 22

40 130]. These mechanical links then influence the survival and proliferation of cells by modulating the activation of growth factor receptors such as TGF-β receptor and epidermal growth factor receptor (EGFR) [ ]. They also modulate the activation of numerous Ca 2+ channels such as TRPV4 [133]. The interaction between chondrocyte and extracellular matrix components via chondrocyte-produced integrins has been shown to mediate the homeostasis and proliferation of articular cartilage [134]. Several types of integrins are produced by chondrocytes, including α1β1, α2β1, α3β1, and αvβ3, and among them, α1β1 and α2β1 are regarded as major collagen VI receptors [130, 135]. Fibronectin is another glycoprotein present in the PCM that aids in cell-adhesion of both human and canine chondrocytes [128, 136, 137]. At the cell surface, fibronectin can bind to integrin α5β1, a fibronectin-specific integrin, as well as to non-integrin receptors such as cellsurface proteoglycans and gangliosides [138]. Both in vivo and in vitro studies have shown an elevated level of fibronectin synthesis in osteoarthritic canine articular cartilage [139, 140]. Similar results were observed in early human OA articular cartilage in vitro, but the mechanism behind this increase is yet to be elucidated [141] The Impact of Osteoarthritis on Articular Cartilage Though OA impacts all joint tissues, its effects are the most prominent on articular cartilage. In general, the progression of OA in articular cartilage occurs in three inter-related stages: changes in matrix structure and composition, chondrocyte response, and finally the loss of tissue [85]. In the initial phase, significant changes are observed in the matrix macromolecular framework, including loss of collagen network integrity and alterations in proteoglycan structure 23

41 (i.e. smaller GAG chains) [ ]. Despite the decrease in swelling pressure due to the loss of aggrecans, higher water content is found in early OA articular cartilage compared to healthy tissue [ ]. Hydration of cartilage is a result of the balance of swelling pressure exerted by the proteoglycans and resistive force provided by the collagen network [145]. Maroudas et al. (1973) hypothesized that damage to the collagen network due to OA results in a reduction of resistive forces that is greater than the reduction of swelling forces due to the loss of proteoglycans, and thus leads to a higher water content in OA cartilage [142]. These early structural changes in the matrix are related to the significant decrease in tissue stiffness and therefore make the tissue progressively vulnerable to further degradation [122, 146]. Additionally, loss of matrix integrity leads to fibrillations of the articular surface of the superficial zone in early OA cartilage [85]. The second phase of cartilaginous change involves the chondrocyte s detection of alterations in electrical, physiochemical, and mechanical stimuli and its response. The cells may respond by proliferating and producing both anabolic and catabolic cytokines to induce cartilage turnover, and this may be the chondrocyte s attempt to repair the tissue [147, 148]. Histologic assessments of osteoarthritic cartilage show clusters of cells, that are indicative of cell proliferation, surrounded by dense matrix macromolecules [147, 148]. Perhaps to accommodate the binding of proliferating clusters of chondrocytes to their matrix, an increase in the deposition of type VI collagen in the pericellular region is also noted during the early phase of OA [106]. Furthermore, the chondrocytes undergoing repair are found to have upregulated expression of anabolic cytokines, such as insulin-like growth factor 1 [149]. When taken together, this suggests that anabolic cytokines play a significant role in cell proliferation and matrix macromolecule synthesis. Upon sensing the stress, chondrocytes are also known to produce nitric oxide which 24

42 diffuses readily through the matrix and stimulates chondrocyte release of IL-1, a catabolic cytokine involved in the breakdown of the matrix [150, 151]. For matrix turnover to occur, fibrillated or damaged matrix must be degraded in addition to the synthesis of the new matrix, and thus the release of both catabolic and anabolic cytokines may be necessary. The repair response of chondrocytes may stabilize, or in some cases restore, the tissue [85]. However, a failed attempt at restoration leads to the last phase of OA, characterized by a gradual loss of articular cartilage. At this stage, what may have begun as a minor articular surface fibrillation becomes a cleft, and the cleft leads to a full-thickness cartilage tear. This progressive loss of tissue integrity may be due to the decreased cellular response to anabolic stimulus, decreased cell activity, and cell death [147]. The full-thickness loss of articular cartilage results in bone-on-bone contact within a joint, which is typically known as one of the primary causes of pain in OA. 1.3 Integrin α1β1 and Osteoarthritis One of the major mechanisms of chondrocyte adhesion to extracellular matrix is through the interaction between integrins and collagen VI. As mentioned previously, collagen VI is primarily concentrated in the PCM and chondrocytes have been revealed to preferentially attach to collagen VI via integrin α1β1 [152]. Collagen VI also binds to integrin α2β1, however its binding affinity was found to be negligible compared to that of the bond between integrin α1β1 and collagen VI [153]. Specifically, integrin α1β1 has been shown to facilitate the 3-d collagen signalling responsible for the expression of matrix metalloproteinase-13, which actively assists in remodelling of both fetal and osteoarthritic cartilage [154]. Ekholm et al. (2002) found that the 25

43 proliferation rate of mesenchymal stem cells (MSCs) was slower in integrin α1-null mice than WT mice [155]. Since MSCs are able to differentiate into bone, cartilage and other connective tissues, MSCs may be critical in cartilage repair, and thus the decreased availability of MSCs in α1-null mice may deprive the tissue of its capacity to repair [156]. Furthermore, Zemmyo et al. (2003) reported that OA expands the site of integrin α1 subunit expression from the growth plate and deep zone of articular cartilage to the superficial and upper mid zones in the murine knee [157]. When taken together, these studies may suggest that integrin plays a critical role in the development and the progression of OA Integrin α1β1 and Spontaneous Osteoarthritis The influence of integrin α1β1 on OA is further supported by the finding that integrin α1- null mice demonstrate an earlier onset of osteoarthritic signs compared to WT, which include severe glycosaminoglycan loss and synovial proliferation [157]. The authors linked these osteoarthritic signs in integrin α1-null mice to an increased rate of cell apoptosis, reduced cellularity, and increased expression of matrix metalloproteinases 2 [157]. Despite the interesting observations made by Zemmyo et al. (2003), they did not consider the importance of sex differences present in the response to the absence of integrin α1β1 [157]. Several studies have demonstrated a significant sex difference in both spontaneous and posttraumatic OA [ ]. For example, Clark et al. (2010) showed that there is a significant sex difference in OA development in TRPV4-deficient mice, such that cartilage wear and bone quality changes are much more prominent in males than their female counterparts [159]. Analyzing histological data without the separation of sex may therefore lead to inaccurate interpretation of the changes induced by the absence of integrin α1β1. Furthermore, Clark et al. 26

44 (2010) also reported significant morphological changes in the bone occurring in conjunction with the histological changes in TRPV4-deficient mice, which Zemmyo et al. (2003) did not consider in integrin α1-null mice [159]. The preliminary data from our laboratory, in which three mice of each genotype was assigned to 6, 8, 10, and 12 month age groups, supports the hypothesis that integrin α1-null male mice develop spontaneous OA earlier and more severely than the WT mice in both bony and soft tissues of the knee, analyzed using micro-computed tomography and histology [161]. In fact, the first sign of spontaneous OA observed in integrin α1-null mice was an increase of subchondral bone density at 10 month of age, 2 months earlier than any soft tissue changes were observed [161]. Other bony changes included increase in calcified meniscal bone volume, increase in subchondral bone thickness in the medial side, and osteophyte growth in integrin α1-null mice (Figure 1.5) [161]. Interestingly, the genotypic differences in soft tissue occurred at an older age (12 months of age, Figure 1.6) than those reported by Zemmyo et al. (2003) who describe significantly greater soft tissue degradation in integrin α1-null compared to WT mice at 7-10 months of age, prior to returning to similar levels of severity at months of age [157]. The difference in the timing of soft tissue degradation between these studies may be due to the different histological scoring approaches used. Neither of these studies addressed the behavioural changes associated with pain resulting from joint damage. The timing and the severity of pain analyzed together with bony and soft tissue changes would allow for comprehensive and clinically relevant characterization of the effect of integrin α1β1 on OA. 27

45 A B Modified Mankin Score WT α1k O Age (Months) # Figure 1.5 Spontaneous OA in integrin α1-null (α1ko) mice is apparent in the soft tissues of the knee at a younger age than in wild-type (WT) controls. (A) 40x (large) and 200x (small) images of a sagittal plane in the centre of the medial condyle of a typical 6 month (6M) and 12 month (12M) old WT and α1ko mouse knee. Sections stained with hematoxylin (nuclei black), fast green (collagen blue) and safranin-o (proteoglycans - pink). Scale bar 40x = 500 µm, 200x = 100 µm. (B) Graph of local histological score (maximum = 30) as a function of age and genotype. Each data point represents the weighted mean of three independent samples. Error bars represent 95% confidence interval, p<0.05. # = significantly different from all data points. 28

46 A B C D Sesamoid Total Volume (mm 3 ) WT α1ko # # Age (Months) E Figure 1.6 Spontaneous OA in the knees of integrin α1-null (α1ko) mice involves subchondral bone thickening and osteophyte development. (A) µct images of 6 month (6M) or 12 month (12M), wild-type (WT) or α1ko murine knees. In frontal images trabecular bone is shaded pink, subchondral bone blue and ossified menisci green. Extensive ossification is indicated in the patellar tendon (arrows) and menisci (arrow heads) in α1ko compared to WT mice. Sagittal images are from the centre of the medial condyle, with a thickness map representing thicker regions with hot colors. (B,C,D,E) Graphs of subchondral bone density (B), trabecular apparent bone density (C), sesamoid total volume (D) and ossified meniscal volume (E) as a function of age and genotype. Each data point represents the weighted mean of three independent samples. HA = hydroxyapatite. Error bars represent 95% confidence intervals, p<0.05. # = significantly different from corresponding WT * = significantly different from 10 month of same genotype. 29

47 1.3.2 Integrin α1β1 and the Potential Mechanisms of Osteoarthritis The mechanism behind the protective role of integrin α1β1 against spontaneous OA is not yet identified. As one of the major mediators between the chondrocyte and its PCM, integrin α1β1 provides the cell with mechanical and biochemical feedback from its environment. The feedback often involves anabolic and catabolic cytokines that subsequently allow the cell to maintain its synthetic and degradative activities, and thus it has the potential to play an important role in the early phases of OA Transforming Growth Factor β Receptor Signalling TGF-β is an anabolic factor that has been shown to play a role in matrix production by chondrocytes in vitro, and the inhibition of endogenous TGF-β reducesh the capacity of cartilage to repair [162, 163]. Interleukin-1, on the other hand, is a degradative factor known to counteract the effects of TGF-β [164, 165]. Interleukin-1 degrades proteoglycan structure in articular cartilage, and the intra-articular injection of IL-1 receptor antagonist is found to reduce the degradation of cartilage in an ACLT model of PTOA [166, 167]. As membrane-residing receptors, integrin α1β1 can interact with and modulate a variety of cytokine receptors, such as IL-1 and TGF-β receptors [132, 168]. Interestingly, Chen et al. (2014) recently reported that integrin α1β1 is essential for the dephosphorylation of type II TGF-β receptor via a mechanism involving T cell protein tyrosine phosphatase (TCPTP) [169]. Parekh et al. (2014) sought to show whether the protective mechanism of integrin α1β1 against OA involves the regulation of chondrocyte responses to IL-1 and TGF-β [170]. They hypothesized that in situ integrin α1-null chondrocytes would have increased sensitivity to IL-1 and decreased sensitivity to TGF-β, contributing to the early onset of spontaneous OA in integrin 30

48 α1-null mice knees. Surprisingly, their findings indicate that chondrocyte intracellular calcium transient responses to IL-1 are down-regulated, but the responses to TGF-β are upregulated in integrin α1-null mice [170]. Despite the assumed protection afforded by the chondrocytes decreased sensitivity to IL-1 and increased sensitivity to TGF-β, integrin α1-null mice still develop early spontaneous OA. However, this assumption of the protective effect of upregulated TGF-β may not be the case. Two studies have shown that prolonged exposure to a high level of TGF-β via adenoviral vector mediated overexpression or multiple intra-articular injections of TGF-β could lead to OA-like signs in the murine knee [171, 172]. This suggests that the lack as well as an overabundance of TGF-β signalling may trigger OA-like signs in joints Epidermal Growth Factor Receptor and Osteoarthritis Epidermal growth factor receptor is a tyrosine kinase receptor that resides on the membrane of a cell, and its overexpression is associated with numerous types of cancers [ ]. Chen et al. (2007) showed that integrin α1β1 down-regulates the EGFR signalling pathway by positively regulating the expression of caveolin-1, a scaffolding protein that controls the signalling of growth factor receptors, and thus suggests a potential involvement of EGFR in the protective mechanism of integrin α1β1 against spontaneous OA [131, 176]. The literature regarding EGFR signalling is primarily focused on cancer and only recently has research been performed on its role in OA. Epidermal growth factor receptor signalling was shown to inhibit chondrogenesis in vitro and impair the cartilage structure by suppressing the production of proteoglycans [ ]. Appleton et al. (2007) reported that there is an increase in the levels of transforming growth factor α (TGF-α), a ligand for EGFR, and EGFR signalling in cartilage of rats 28 days following a surgical induction of OA (ACLT and partial medial 31

49 meniscectomy) [181]. In addition, they also showed that the TGF-α transcript levels are increased in a subset (5 out of 12) of human knee OA patients, suggesting an increased level of EGFR signalling in human OA articular cartilage as well [181]. In 2005, Zhang et al. reported that targeted disruption of a mitogen-inducible gene 6 (mig- 6), which normally inhibits the activation of EGFR, led to the early onset of OA detectable at 4 months of age [ ]. Surprisingly, an early (6 weeks of age) anabolic response to EGFR activation was observed in mice with mig-6 conditional loss targeted to the limbs, which included significant thickening of cartilage and three to four times higher cell proliferation [186]. However, the prolonged activation of EGFR in the conditional knockout mice led to the breakdown of cartilage, a catabolic response similar to the results of another Zhang et al. (2005) study [182, 186]. Interestingly, transgenic mice with a mutated EGFR, and thus with the lack of EGFR signalling, were also shown to have increased cartilage destruction [187]. Together, these studies suggest that the role of EGFR is similar to that of TGF-β in that both the lack and the abundance of either protein induce an abnormal chondrocyte response that ultimately results in the OA-like degradation of joints. The inhibition of EGFR signalling can also be achieved pharmaceutically. Erlotinib, also referred to as Tarceva, is an EGFR inhibitor that is approved by the Food and Drug Administration agency for use in various cancer treatments [188]. In one study related to arthritis, the oral administration of erlotinib was found to significantly reduce the severity of collageninduced rheumatoid arthritis [189]. Gefitnib is an alternative drug that has a different structure and toxicity to erlotinib but performs a similar function [190]. To our knowledge, no studies looked at the effects of gefitnib on arthritis prior to the year Very recently, a study showed that the administration of gefitnib to male mice with induced PTOA for 12 weeks resulted in 32

50 slightly more severe OA like changes in femoral condyle compared to vehicle-administered mice, albeit with a small sample size (8 vehicle and 9 treatment) [187]. Thus far, the effect of erlotinib on spontaneous or post-traumatic OA in mice has not been elucidated. 1.4 Rationale Integrin α1β1 delays the onset and reduces the severity of spontaneous OA however the role of integrin α1β1 in PTOA has not been studied. A review paper comparing the different models of OA in genetically-modified mice reported that the majority (10/15) of the genetic modifications had the same effect on both spontaneous OA and PTOA [191]. However, this author also showed that in five other genetically-modified strains, the effect of genetic modification was different, or even opposite, in these contrasting models of OA [191]. This suggests that the effects of a genetically-modified strain may not necessarily be the same in different models of OA and thus it is important to also understand the function of integrin α1β1 on PTOA. In addition, generating knockout and congenic strains that have 99% similarity in background takes at least 6 to 10 generations, and this alone may take two or more years [192, 193]. Furthermore, the additional time it takes in aging the mice and the large sample size required due to the variability of the induction make spontaneous models costly and time consuming to conduct relative to their PTOA cousins [192]. Many animal models of PTOA have been established, including ACLT and destabilisation of medial meniscus (DMM) [192, 194]. In comparison to the DMM model, the ACLT surgery is a more common model of PTOA in many animals (i.e. Dogs, cats, and sheep) due to it mimicking a more naturally occurring injury in humans. However, in mice, the ACLT surgery has been shown to result in exacerbated signs of OA that do not resemble those of spontaneous 33

51 OA in mice [192]. In comparison, the DMM surgery produces moderate to severe OA-like signs in a span of 8 weeks, allowing enough time to study its progression [192]. ACLT leads to global joint instability that perhaps exceeds what the small mouse joint can bear, whereas DMM only produces instability in the medial compartment of the joint with a relatively lower stress, and hence slowing the progression of the disease. Additionally, ACLT may lead to more iatrogenic damage and bleeding within the synovial cavity in a small joint, a known risk factor for OA [192, 195]. Therefore, the use of the DMM model is recommended over ACLT in mice [192]. The DMM model has often been used in the literature, and the behavioural changes in mice and morphological changes in soft and bony tissues of the knee post-surgery have been well characterized in WT mice [192, ]. This allows for comparing and contrasting the results of this study to determine the validity and accuracy of the procedure performed. More importantly, the DMM model has already been used to validate another potential pharmaceutical target for OA called ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin-like motifs 5) in gene knockout mice, suggesting that it may be a suitable model to detect any protective effects of integrin α1β1 against OA [199, 200]. As far as we are aware, no study has performed the DMM procedure on integrin α1-null mice to date. Integrin α1β1 affords protection to the knee against spontaneous OA however the mechanisms by which it performs this task are currently unknown. Two potential mechanisms involve down-regulation of TGF-β and/or EGFR signalling pathways via the interplay of integrin α1β1 and TGF-β receptor and EGFR respectively. Generally, TGF-β is considered as an anabolic factor involved in restoration of the tissue damage, but an overabundance of TGF-β signalling, such as in the case of integrin α1-null mice, was shown to aggravate the signs of OA [162, ]. Similarly, integrin α1-null mice are shown to have a high level of EGFR signalling, which 34

52 is also associated with early onset and severe signs of spontaneous OA [176, 182]. In this thesis we will investigate the role of EGFR signalling in the differential development of PTOA in integrin α1-null and WT mice. Based on the literature presented, we hypothesize that: 1. The effect of PTOA, characterized by loss of cartilage, growth of osteophytes, and decreased activity levels, would be exacerbated in integrin α1-null mice at every time point compared to WT mice. 2. The protective mechanism of integrin α1β1 against PTOA involves the dampening of EGFR activation. To test these hypotheses, we will perform DMM surgery on integrin α1-null and WT mice and monitor the progression of PTOA over 12 weeks using micro-computed tomography, histology, and behavioural testing. Furthermore, we will administer erlotinib to test whether EGFR signalling is one mechanism by which integrin α1β1 protects the knee from PTOA. As such, the specific aims of this study are: 1. To quantify changes in the calcified tissues of the knee using micro-computed tomography, and changes in articular cartilage and synovium using histological methods, in integrin α1-null and WT mice at 2, 4, 8 and 12 weeks post DMM surgery. 2. To quantify changes in withdrawal threshold, grip strength, stride length, and activity levels in male integrin α1-null and WT mice at 4, 8 and 12 weeks post DMM surgery. 3. To administer erlotinib to an additional group of integrin α1-null and WT mice from the day after DMM surgery until sacrifice at 12 weeks post-surgery to elucidate the role of EGFR signalling as a mechanism by which integrin α1β1 protects against PTOA. 35

53 Chapter Two: Integrin α1β1 Protects Against Signs of PTOA in the Articular Cartilage of the Knee Through a Mechanism that Involves EGFR Signalling 2.1 Introduction Osteoarthritis (OA) is a degenerative joint disease affecting 1 in 10 Canadians [201]. It typically manifests as fissured and then degraded articular cartilage, thickened subchondral bone, osteophyte growth at the joint margins and synovitis [1]. For soccer players, the risk of developing post-traumatic OA (PTOA) after an anterior cruciate ligament tear is 78% within 14 years of injury [202]. In contrast to spontaneous OA where diagnosis normally occurs with endstage disease when patients present with pain, the period of latency between injury and the development of PTOA offers a critical opportunity to intervene in the disease process before any signs or symptoms of OA are apparent. Despite this treatment window, there are still no effective strategies to delay or slow the progression of PTOA. Integrins are heterodimeric collagen receptors that modulate the activation of growth factor receptors such as transforming growth factor β (TGF-β) receptor (TβR) and epidermal growth factor receptor (EGFR) [131, 132]. Integrin α1β1 is present on human chondrocytes [203] and is a major collagen binding receptor responsible for 75% of chondrocyte adhesion to chondron localized collagen VI [204] and 38% of attachment to collagen II [203]. In knee cartilage, spontaneous OA results in an increase in the realm of integrin α1β1 expression from the deep cartilage zone into the superficial zone before macroscopic changes such as cartilage fibrillation or fissuring occur [157, 203]. This timing likely coincides with type VI collagen remodelling in superficial chondrons during OA [120]. Due to the lack of availability of human tissues of early 36

54 OA, these observations have been made in two animal models of spontaneous OA the cynomolgus macaque [203] and BALB/c mice [157]. Interestingly, integrin α1-null mice (mice lacking integrin α1β1 due to the exclusive pairing of the integrin α1 subunit with the β1 subunit) develop spontaneous OA two months earlier than controls, with OA signs apparent in both bone and soft tissues of the knee [157, 161]. The mechanism(s) by which integrin α1β1 delays the signs of spontaneous OA and the role of integrin α1β1 in PTOA, are unknown. Two potential protective mechanisms of integrin α1β1 against OA may involve down-regulation of EGFR and/or TβR signalling pathways via the interplay of integrin α1β1 with these growth factor receptors. Briefly, integrin α1β1 negatively regulates both EGFR and TβR signalling in a ligand independent manner involving activation of the phosphatase TCPTP. Thus integrin α1-null mice experience enhanced EGFR and TβR signalling [169, 170, 176, 205]. Epidermal growth factor receptor is a tyrosine kinase receptor activated by the EGF family, including TGF-α [206, 207]. Expression levels of TGF-α in the synovium, synovial fluid and cartilage are increased in patients with OA [ ]. In 2005, Zhang et al. reported that targeted disruption of mitogen-inducible gene 6 (mig-6), an inhibitor of EGFR, led to the early onset of spontaneous OA detectable at 4 months of age [182]. Since then, more severe spontaneous OA as a consequence of enhanced EGFR activation has been confirmed in male and female mice with global or cartilage-specific deletion of mig-6 [212, 213]. Recently, however, a contrasting effect of EGFR signalling in PTOA has been reported by Zhang et al. (2014). They showed enhanced cartilage damage post destabilisation of medial meniscus (DMM) in male mice with dampened EGFR signalling achieved by genetic mutation of EGFR or the drug gefitinib [187]. Importantly however, the influence of sex and tissue type (bone vs. cartilage) on the effects of dampened EGFR signalling on PTOA remains unknown. 37

55 Generally, TGF-β is considered an anabolic factor involved in restoration of tissue damage, however an overabundance of TGF-β signalling in the mouse knee, through multiple injections of TGF-β1 [165, 214] or the overexpression of active TGF-β1 [171], leads to chondrophytes that develop in both ligament, meniscal and cartilaginous tissues four days post treatment as well as proteoglycan depletion in tibial cartilage that progresses to lesions indicative of mouse OA at 2 months of age [165, 171, 214]. In light of these data, a degradative effect of elevated TβRmediated signalling that contributes to the early OA experienced by integrin α1-null mice cannot be ruled out. The goal of this study was to examine the role of integrin α1β1 in the progression of PTOA. We hypothesized that the effect of PTOA, characterized by synovitis, loss of cartilage and growth of osteophytes, would be exacerbated in α1-null mice at every time point post DMM surgery compared to wildtype (WT) mice. Furthermore, we administered erlotinib, an EGFR inhibitor approved by the Food and Drug Administration to treat non-small cell lung cancer, to test whether dampened EGFR signalling is one mechanism by which integrin α1β1 protects the knee from PTOA. 2.2 Materials and Methods Animals All methods were approved by the University of Calgary Animal Care Committee. Hundred α1-null and 100 WT BALB/c mice, half male and half female mice, were used in this study after verification of genotype by polymerase chain reaction of ear punch tissue (Appendix 38

56 A) [215]. Mice were exposed to daily 12-hour light/dark cycles and were provided free access to food and water throughout Surgery At 13±1 weeks of age, mice were placed under anesthetic (isoflurane), hair removed from the left leg using clippers (Model 5540; Wahl Clipper Corp., Sterling, IL) and skin sterilised with 0.5% chlorhexidine (Partnar Animal Helath Inc., Ilderton, ON). Buprenorphine (Buprenex, Recklitt and Coleman Products, Kingston-Upon-Hull, UK) was administered subcutaneously at 0.05mg/kg. Microsurgery to destabilise the medial meniscus (DMM) or sham surgery was then performed on the left knee [192]. After surgery, the joint capsule was closed using 8-0 tapered Vicryl suture, and a single 9mm staple applied to close the skin. Immediately following surgery, mice were weighed and placed in a recovery cage for close observation until full recovery. The staple was removed one week after surgery. Hundred and twenty mice, with equal numbers of surgery type (DMM/sham), genotype (α1-null/wt), and sex (male/female), were assigned to each of the 2, 4, and 8 weeks post-surgery (PS) groups. Eighty mice with the same proportions as above were assigned to the 12 weeks PS group Erlotinib Administration Mice in the 12 weeks PS group received 50 mg/kg/day erlotinib (Genentech, San Francisco, CA) suspended in 0.5% (w/v) hydroxypropyl methyocellulose (Sigma-Aldrich, St. Louis, MO) (Dow) and 0.1% (v/v) Tween 80 in distilled water or vehicle by oral gavage (Appendix B). Erlotinib hydrochloride (Tarceva ) is an EGFR inhibitor whose specificity to EGFR is greater than that of the tyrosine kinases v-abl and c-src and other kinase domains including that of the 39

57 insulin receptor and insulin-like growth factor 1 receptor [216, 217]. A two-year carcinogenicity study at oral dose of 60 mg/kg/day revealed no carcinogenic or mutagenic effects in mice [188]. Gavage began the day after surgery and continued until the day before euthanization Euthanization and Tissue Preparation At their assigned time point PS, mice were euthanized (CO2), blood collected via cardiac puncture and hindlimbs removed using microdissection (Appendix C). Skin was removed and the foot excised at the ankle and the femur at midshaft (Appendix C). The limbs were then stored submerged in phosphate buffered saline (PBS) ph 7.4 (Invitrogen) in cryotubes at -80 C until further processing. Contralateral knees of DMM joints were used as a naïve control Micro-Computed Tomography Hindlimbs were thawed and fixed for 48 hours at a physiological joint angle in 10% neutral buffered formalin (RICCA, Arlington, TX). In a single scan, four hindlimbs were held vertically in a cylinder-shaped foam, approximately equidistance from one another, and were carefully positioned into the formalin-filled scanning cylinder (Appendix D). The limbs were aligned such that the heights of the joint space of all four limbs were approximately equal. The cylinder was then placed inside the micro-computed tomography (microct) machine (microct 35; Scanco Medical, Brüttisellen, Switzerland) for scanning, and high intensity medium resolution (16µm) microct scans in the transverse plane were obtained. Calibration of the microct machine was performed on a weekly basis using a phantom containing five rods of known hydroxyapatite densities to ensure consistent image and density measurements [218]. 40

58 Four regions of subchondral and trabecular bone (medial and lateral tibial plateaus and femoral condyles), the calcified anterior and posterior horns of medial and lateral menisci, the medial and lateral fabella [219] and the calcified portions of the medial collateral ligament were analysed (Figure 2.1A). The subchondral region of each condyle was defined from the first transverse slice where the subchondral bone appeared to the twelfth slice above or below, for femoral and tibial bone, respectively (Appendix E). The trabecular region of femur and tibia were defined as the first slice where trabeculae appeared through to the slice just prior to the appearance of the growth plate. A semi-automated contouring tool and a custom written code was used to define the regions of interest and evaluate total volume, bone volume, bone volume fraction, bone mineral density, and tissue mineral density for all regions (IPL; Scanco Medical AG, Brüttisellen, Switzerland). In addition to the above, connectivity density, structure model index (SMI), trabecular thickness, and trabecular spacing were evaluated for trabecular bone regions. For subchondral bone, its thickness was measured using a circle-drawing algorithm where circles of maximum diameters are fitted into the subchondral plate (Figure 2.1D). Because the size of the circle diameter depends on the dimension of the subchondral bone, the measurement of its diameter represents the subchondral bone thickness (Appendix F). Three thickness measurements were taken at points evenly spaced across the load bearing region of each condyle and plateau in the coronal plane. The load bearing region was defined as the coronal section of the joint where the joint space was minimal in both the medial and lateral compartments. The average of the three measurements was calculated for each condyle and plateau of each specimen. The calcified medial collateral ligament (cmcl) was scored by finding the range of bone volume and tissue mineral density across all cmcl, then scoring each joint a score out of 3 for bone volume and 3 for tissue mineral density relative to that range (0 = 41

59 no cmcl, 1 = bottom third, 2 = middle third, 3= top third). The two scores were added together for a maximum score of 6 per joint, and the maximum score of each group was reported. Figure 2.1 Representative microct images of left female and male murine knees at 2 and 12 weeks post-surgery (PS) that underwent either destabilisation of the medial meniscus (DMM) or sham surgery. Mice in the 12 weeks PS group were administered erlotinib (12E) or vehicle (12) daily. A-C, E-G, I-K, M-O Cortical bone at the femoral condyles and tibial plateaus is rendered transparent to show the analyzed trabecular regions. Trabecular bone is shaded blue, subchondral bone pink, and ossified menisci green. D, H, L, P Frontal cross-section taken in the middle of femoral condyle/tibial plateau contact region shaded with a thickness map with thicker regions shaded with hot colors located further right of the spectrum scale. Note the medial displacement of medial meniscus in DMM joints (A-C, I-K), larger medial meniscus and calcified medial collateral ligament at 12 weeks PS in DMM knees (B, J) with this effect lessened with erlotinib treatment in female (C) but worsened in male mice (K), and thicker subchondral bone in DMM (D, L) compared to sham (H, P) joints. 42

60 2.2.6 Histology After microct scanning, hindlimbs were dissected free of muscle (leaving the knee capsule intact) and then decalcified (Appendix D). After dehydration and paraffin embedding, coronal histological sections 8 μm thick were cut throughout the entire knee [159]. During cutting, the block was occasionally moistened with wet gauze to prevent drying of the tissue. Sections on glass slides were stained with hematoxylin, fast green and safranin-o and coverslipped [159]. Eight sections at μm intervals per joint in the center of each contact region were imaged at 40x (Zeiss AxioCam ICc1; Jena, Germany) and were scored by three trained and blinded graders using the Osteoarthritis Research Society International grading scale (Figure 2.2) [220]. The sum and maximum histological score across the eight sections per joint at each of the four regions (medial and lateral tibial plateaus and femoral condyles) were calculated separately. Synovitis was scored based on the enlargement of the synovial lining cell layer (0 = 1-2 cells thick, 1 = 2-4 cells thick, 2 = 4-9 cells thick, 3 = Thickness 10 cells, modified from [221]) at all four regions of the joint. The scores of all four regions were summed and the maximum score of each group was reported Statistical Analysis The data were consolidated and managed electronically in Excel (Microsoft, Redmond, WA). Statistica (StatSoft Inc., Tulsa, OK) was used for all statistical analysis. Statistical significance was defined as p<0.05. All data are presented as mean ± 95% confidence interval. All histological cartilage and morphological bone data were analyzed using Multivariate analysis of variance (MANOVA) with all applicable independent categorical variables (time PS (2, 4, 8, and 12) weeks), sex (male/female), genotype (α1-null/wt), surgery type (DMM/sham), drug 43

61 (erlotinib/vehicle) bone (femur/tibia), site (anterior/posterior), and compartment (medial/lateral) and a continuous predictor of mass. The Fisher LSD post hoc test was performed to determine significance. Statistical analyses of the frequency of cmcl and synovitis were performed using chi-square and t-tests. Figure 2.2 Representative frontal histology images of male and female α1-null and wild type (WT) left knees at 2, 4, 8 or 12 weeks post-surgery (PS). Knees underwent either destabilisation of the medial meniscus (DMM) or sham surgery and mice in the 12 weeks PS group were administered erlotinib (12E) or vehicle (12) daily. Sections were stained with hematoxylin, fast green, and safranin-o. Scale bar represents 500µm. Note the thinner and more fibrillated cartilage in the medial compartment (arrows) beginning at 4 weeks PS in male α1-null and WT DMM knees with this effect delayed until 8 weeks PS in female α1-null mice and 12 weeks PS in female WT mice. Erlotinib treatment alleviated cartilage damage in both male and female α1- null but not WT mice. 44

62 2.3 Results Animals Male mice weighed approximately 5g more than female mice and had consistent weight across the time point groups at surgery (Figure 2.3A). Figure 2.3 Mouse mass at surgery (A) and change in mouse mass from surgery to sacrifice (B) as a function of time post-surgery (PS, weeks), sex and genotype. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 9) ± 95% confidence interval. a Different from 8, 12, and 12E weeks PS equivalent (P < 0.05). b Different from 12E weeks PS equivalent (P < 0.05). c Different from 4 and 8 weeks PS equivalent (P < 0.05). d Different from all points equivalent (P < 0.05). e Different from 2, 12, and 12E weeks PS equivalent (P < 0.05). Female α1-null mice in the 2 and 4 weeks PS group were 2g heavier than the equivalent mice assigned to later time point groups, and female erlotinib treated WT mice weighed 4g less than their vehicle treated counterparts (Figure 2.3A). All mice in the 8 weeks PS group gained significantly more mass during the study compared to mice assigned to all other time points, and in WT mice of both sexes an increase in mass was evident in the 4 week compared to 2 weeks PS group (Figure 2.3B). Interestingly, there was minimal change in mass in all 12 and 12E group 45

63 mice, suggesting that the weight gained at other time points was due to food consumption rather than skeletal growth (Figure 2.3B). All DMM surgeries were confirmed after sacrifice by observing a medially displaced medial meniscus in microct images (Figure 2.1A-C and I-K). One 8 weeks PS WT male mouse that underwent sham surgery was identified as having undergone DMM surgery after sacrifice and was reassigned to the equivalent DMM group. One sham WT female mouse assigned to the 12 weeks PS group and receiving erlotinib was found dead in her cage at 7 weeks PS and was excluded from the study. Four additional female mice assigned to the 12 weeks PS group and receiving erlotinib treatment demonstrated signs of hydrocephalus including an enlarged, domed head, dehydration and depression close to their assigned time point. As recommended by animal care staff, one of them (WT DMM) was sacrificed at 10 weeks PS and three others (α1-null sham) were sacrificed at 11 weeks PS. Their data were included in the study Subchondral Bone The subchondral bone was thicker at 8 and 12 weeks PS in DMM compared to sham and contralateral joints in both femur and tibia, but only in the medial compartment (Figure 2.1D, H, L, P and Figure 2.4A-D). Interestingly, the thickened subchondral bone after surgery was diminished in erlotinib treated females in both the femur and tibia (Figure 2.4A, B) but increased in males in the femur (Figure 2.4A). In addition to thickness, DMM surgery significantly affected subchondral bone tissue mineral density and bone volume (Figure 2.5) with both parameters being larger in DMM compared to sham and contralateral joints in the medial femur. 46

64 Figure 2.4 Subchondral bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of (A, B) sex and bone or (C, D) sex and compartment. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean n 9 ± 95% confidence interval. a Different from 2, 4, and 12E weeks post-surgery (PS) equivalent (P < 0.01). b Different from all time points PS equivalent (P < 0.05). c Different from 2 and 12E weeks PS equivalent (P < 0.05). d Different from 2 and 4 weeks PS equivalent (P < 0.01). e Different from 12 weeks PS (P < 0.05). f Different from 2, 4, and 8 weeks PS (P < 0.05). g Different from 4, 8, and 12E weeks PS equivalent (P < 0.05). h Different from 8, and 12E weeks PS equivalent (P < 0.05). * Different from time-matched counterpart(s) (P <0.05). Note increased thickness with DMM in both femur (A) and tibia (B) but only in the medial compartment (C), with this diminished in erlotinib treated females but not males (A-C). 47

65 Figure 2.5 Subchondral bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of compartment and bone. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (A, B n 19, C n 99, D n 39) ± 95% confidence interval. a Different from 2 and 4 weeks PS equivalent (P < 0.01). b Different from 2, 4, and 12E weeks post-surgery (PS) equivalent (P < 0.01). c Different from 2 and 12E weeks PS equivalent (P < 0.05). d Different from 4, 8, and 12E weeks PS equivalent (P < 0.05). e Different from 8, and 12E weeks PS equivalent (P < 0.05). f Different from all points (P < 0.05). g Different from contralateral equivalent (P < 0.05). * Different from surgery- or time-matched counterpart(s) (P <0.05). Note (A) increased density with DMM in the medial compartment of the femur reduced with erlotinib treatment, (C) increased bone volume with DMM in both femur and tibia but only in the medial compartment, and (D) a contrasting effect of erlotinib in the medial and lateral compartments of the tibia but not the femur. 48

66 Erlotinib treatment diminished the increase in tissue mineral density with surgery in both sham and contralateral knees but the decrease did not reach statistical significance in DMM joints (Figure 2.5A). Finally, subchondral bone volume was larger in the medial than the lateral compartment, and erlotinib treatment decreased this parameter in the medial compartment but increased it in the lateral compartment, but only in tibia (Figure 2.5C, D) Calcified Meniscus Calcified meniscal bone volume was larger in DMM joints at 8 and 12 weeks PS compared to sham and contralateral joints at both anterior and posterior sites (Figure 2.6A, B) but only in the medial compartment (Figure 2.1B, F, J, N and Figure 2.6C). Erlotinib treatment diminished this surgery effect in females at both anterior and posterior sites but had no affect or increased the surgery effect in males at the posterior and anterior sites respectively (Figure 2.1C, G, K, O and Figure 2.6A, B). When female and male data were combined, a significant drug effect was observed in the medial but not the lateral compartment of the knee (Figure 2.6C) Calcified Medial Collateral Ligament More than twice as many females had a calcified medial collateral ligament (cmcl) compared to males, but the severity of calcification (as measured by bone volume and density) was similar between sexes (Table 2.1). In a similar manner, approximately twice as many DMM joints had a cmcl compared to sham and contralateral joints, but with similar severity across surgery groups (Figure 2.1B, F, J, N and Table 2.1). The effect of surgery on increased frequency of cmcl was seen at 8 weeks PS (Table 2.1). No genotype or drug effects on cmcl were observed (Table 2.1). 49

67 Figure 2.6 Meniscal bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of (A, B) site and sex or (C) compartment. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (A, B n 9, C n 19), ± 95% confidence interval. a Different from all time points equivalent (P < 0.05). b Different from 2 and 4 weeks PS equivalent (P < 0.01). * Different from time-matched counterpart(s) (P <0.05). Note (A, B) the increase in meniscal bone volume with DMM in both anterior and posterior sites but (C) only in the medial compartment and (A, B) the reduction of this with erlotinib treatment in females but not males. 50

68 Table 2.1. The frequency and severity of calcified medial collateral ligaments (cmcl) in murine knees as a function of time post-surgery, sex, genotype, surgery to destabilise the medial meniscus (DMM), and erlotinib treatment. Calcification was scored for bone volume and density, and summed for a maximum calcification score of 6. Note that approximately twice as many cmcl were observed in females compared to males and in DMM compared to sham or contralateral joints. Significant differences: (p=0.009); (p=0.049); (p=0.006) Time Post-Surgery E Sum Maximum Sum Male Female α1-null Wildtype DMM Contralateral Sham Erlotinib Vehicle # of mice with cmcl Total # of Joints # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score # of mice with cmcl Total # of Joints Maximum Score

69 2.3.5 Fabella Medial but not the lateral fabellae were larger in DMM compared to sham and contralateral joints at all time points PS (Figure 2.7A). Bone volume fraction was also larger in DMM compared to sham joints at 4, 8 and 12 weeks PS and in DMM compared to contralateral joints at 4 and 12 weeks PS (Figure 2.7B). Erlotinib treatment significantly reduced both lateral and medial fabella bone volume and bone volume fraction, with this effect being most consistent in DMM compared to sham and contralateral joints (Figure 2.7A, B). The lateral and medial fabellae were significantly larger, but less dense in male compared to female mice at all time points (Figure 2.7C, D). A 4 week delay in the increase of tissue mineral density in males compared to females was also observed in both lateral and medial fabellae (Figure 2.7D). The effect of Erlotinib treatment was sex dependent, decreasing both bone volume and tissue mineral density in females but not males (Figure 2.7C, D) Trabecular Bone Surgery did not significantly affect any of the trabecular bone parameters. However erlotinib treatment consistently altered multiple parameters of trabecular bone in female, but not male mice (Figure 2.8). Tissue mineral density in both femur and tibia (Figure 2.8A, B), and bone volume fraction (Figure 2.8C) and trabecular thickness (Figure 2.8D) in the femur were all decreased in female mice receiving erlotinib, in contrast to femoral connectivity density (Figure 2.8E) which was increased. Additionally, there was a notable 4 week delay in the increase of femoral tissue mineral density (Figure 2.8A), bone volume fraction (Figure 2.8C), and trabecular thickness (Figure 2.8D) in male compared to female mice. In general, tissue mineral density 52

70 (Figure 2.8A, B), bone volume fraction (Figure 2.8C), and trabecular thickness (Figure 2.8D) were larger and connectivity density (Figure 2.8E) smaller in females compared to males. Figure 2.7 Fabella bone remodelling in murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of (A-D) compartment and (C, D) sex. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 19) ± 95% confidence interval. a Different from 2 and 4 weeks PS equivalent (P < 0.05). b Different from 12E weeks PS equivalent (P < 0.05). c Different from 2 and 8 weeks PS equivalent (P < 0.05). d Different from 8 weeks PS equivalent (P < 0.05). e Different from 2 weeks PS equivalent (P < 0.05). f Different from 12 weeks PS equivalent (P < 0.05). g Different from 12 and 12E weeks PS equivalent (P < 0.05). * Different from time-matched counterpart(s) (P <0.05). Note (A, B) the significant effect of DMM in the medial but not lateral compartment and the reduction of this with erlotinib treatment and (C, D) the significant effect of erlotinib in females but not males, independent of surgery. 53

71 Figure 2.8 Trabecular bone remodelling in murine knees 2, 4, 8, and 12 weeks post-surgery (PS) as a function of sex, compartment and bone. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 19) ± 95% confidence interval. a Different from 2 weeks PS equivalent (P < 0.05). b Different from all other points (P < 0.01). c Different from 8 weeks PS equivalent (P < 0.05). d Different from all other points except 8 weeks PS equivalent (P < 0.01). e Different from 2 and 12E weeks PS equivalent (P < 0.05). * Different from time-matched counterpart(s) (P <0.05). Note the significant effect of erlotinib in females but not males on all parameters and a delayed response in males compared to females in some femoral parameters. 54

72 Bone volume fraction was larger (Figure 2.9B), and trabecular spacing smaller (Figure 2.9D) in the tibiae of WT compared to α1-null mice at 2 and 4 weeks PS. Erlotinib treatment reduced femoral and tibial bone volume fraction (Figure 2.9A, B) in female but not male mice independent of genotype, however erlotinib reduced tibial trabecular spacing (Figure 2.9D) only in WT female (but not male) mice. Figure 2.9 Trabecular bone remodelling in murine knees 2, 4, 8, and 12 weeks post-surgery (PS) as a function of genotype, sex and bone. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. Data points represent mean (n 9) ± 95% confidence interval. a Different from 2 and 12E weeks PS equivalent (P < 0.05). b Different from 4 and 8 weeks PS equivalent (P < 0.05). c Different from 8 weeks PS equivalent (P < 0.05). d Different from 12, and 12E weeks PS equivalent (P < 0.05). e Different from 2, 4, and 12E weeks PS equivalent (P < 0.05). f Different from 8 and 12 weeks PS equivalent (P < 0.01). * Different from time-matched counterpart(s) (P <0.05). Note (A, B) the significant effect of erlotinib in females but not males and (B, D) genotypic differences in tibial parameters in females at early time points. 55

73 2.3.7 Cartilage In male mice of both genotypes, increased cartilage damage was seen as early as 4 weeks PS in the medial compartment of both femur and tibia of DMM but not sham or contralateral knees (Figure 2.2, Figure 2.10&Figure 2.11A, B). Interestingly, this surgery effect was delayed until 8 weeks PS in α1-null females (Figure 2.2, Figure 2.10&Figure 2.11C) and 12 weeks PS in WT females (Figure 2.2, Figure 2.10&Figure 2.11D). Despite this contrasting time of onset in cartilage degradation in male and female mice, the severities of both summed and maximum cartilage degeneration at 12 weeks PS were similar between the two sexes (Figure 2.10&Figure 2.11). Importantly, erlotinib treatment decreased maximum histology score in both sexes (Figure 2.10) and summed histology score in females only (Figure 2.11) primarily in the medial femoral condyle of α1-null but not in WT mice Synovitis The frequency of synovitis was four times higher in DMM compared to sham and contralateral joints, and was observed at all time points PS (Table 2.2). Furthermore, synovitis was present in more α1-null compared to WT mice, though did not reach statistical significance (Table 2.2). Sex and erlotinib had no effect on the frequency of synovitis, and the maximum severity of synovitis was similar across all groups (Table 2.2). 56

74 Figure 2.10 Maximum histology osteoarthritis score (out of 6) of murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of sex, genotype, and region. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. MFC Medial Femoral Condyle. MTP Medial tibial plateau. LFC Lateral femoral condyle. LTP Lateral Tibial Plateau. Data points represent mean (n 4) ± 95% confidence interval. a Different from 2 and 12 weeks PS (P < 0.01). b Different from all points of equivalent region (P < 0.05). c Different from 2, 8, and 12 weeks PS equivalent (P < 0.05). d Different from 2 weeks PS equivalent (P < 0.01). e Different from 2 and 4 weeks PS (P < 0.01). f Different from 2, 4, and 8 weeks PS equivalent (P < 0.01). * Different from time-matched medial compartment (P <0.05). Note the significant effect of DMM in the medial compartment, the erlotinib effect in αl-null but not wildtype mice, and a delayed response in females compared to males and in WT females compared to α1-null females. 57

75 Figure 2.11 Summed histology osteoarthritis score (out of 48) of murine knees 2, 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of sex, genotype, and region. Mice in the 12 week group were administered erlotinib (12E) or vehicle (12) daily. MFC Medial Femoral Condyle. MTP Medial tibial plateau. LFC Lateral femoral condyle. LTP Lateral Tibial Plateau. Data points represent mean (n 4) ± 95% confidence interval. a Different from 2 and 4 weeks PS equivalent (P < 0.05). b Different from 2, 12, and 12E weeks PS equivalent (P < 0.01). c Different from 2, 4, and 12E weeks PS equivalent (P < 0.05). d Different from all points of equivalent region except 12E (P < 0.01). e Different from all points of equivalent region (P < 0.05). * Different from time-matched medial compartment (P <0.05). Note the significant effect of DMM in the medial compartment, the erlotinib effect in αl-null but not wildtype mice, and a delayed response in females compared to males and in WT females compared to α1-null females. 58

76 Table 2.2. The frequency and severity of synovitis in murine knees as a function of time, sex, genotype, surgery to destabilise the medial meniscus (DMM), and erlotinib treatment. Synovitis was scored at all four regions (medial and lateral tibial plateau and femoral condyle) of the joint and summed. The maximum score per joint on this scale is 12. Note the four fold increase in the frequency of synovitis in the DMM compared to sham and contralateral joints, and the increased frequency of synovitis in α1-null compared to the wildtype mice. Significant differences: (p=0.001) Time Post-Surgery E Sum Maximum Sum Male Female α1-null Wildtype DMM Contralateral Sham Erlotinib Vehicle # of mice with Synovitis Total # of Joints # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score # of mice with Synovitis Total # of Joints Maximum Score

77 2.4 Discussion The goal of this study was to examine the role of integrin α1β1 in the progression of PTOA and to test whether dampening EGFR signalling is one mechanism by which integrin α1β1 protects the knee from PTOA. As hypothesized, integrin α1-null mice showed earlier cartilage degradation post DMM surgery compared to WT female, but not male, mice. Additionally, we observed a trend towards a higher frequency of synovitis in α1-null compared to WT mice. Surprisingly however, all of the effects of PTOA on ossified tissues of the knee were independent of genotype. This mildly protective influence of integrin α1β1 on the progression of PTOA contrasts with its more extensive role, albeit still protective, in spontaneous OA that includes cartilage degradation, calcified meniscal volume and the thickness and density of subchondral bone [157, 161]. As we originally hypothesized, the majority (2/3) of genetic modifications in mice have the same effect on both spontaneous and post-traumatic OA, however some demonstrate a diminished effect on one subtype of OA compared to the other as seen here in α1-null mice, or in some cases even an opposite effect [191]. This diminished protective role of integrin α1β1 against PTOA compared with spontaneous OA may be due to the considerable differences between these subtypes of OA, including knee trauma, mechanical instability, and the timeframe of disease progression (weeks vs. months) [157, 161, 192]. In the articular cartilage where the effects of integrin α1β1 on PTOA were most apparent, dampening EGFR signalling through erlotinib treatment resulted in significantly less cartilage damage in α1-null but not WT mice. This evidence supports our hypothesis that the dampening of EGFR signalling by integrin α1β1 may be one mechanism by which integrin α1β1 protects against OA. Further in vitro studies comparing EGFR signalling in α1-null and WT chondrocytes may provide additional evidence to support this hypothesis and shed light on the specific 60

78 pathways downstream of EGFR that are influencing OA progression in the cartilage of α1-null mice. In addition to EGFR, integrin α1β1 also dampens TβR signalling [169] and α1-null chondrocytes have been shown to have a ten-fold lower threshold of TGF-β required to elicit a calcium response compared to WT counterparts, and heightened Smad 2/3 activation downstream [170]. Therefore, heightened TβR signalling in the cartilage of α1-null mice may also be playing a role here in the earlier cartilage degradation seen in α1-null compared to WT mice post DMM surgery. Independent of genotype, dampening EGFR signalling by erlotinib treatment significantly mitigated the effects of PTOA on many tissues of female mice including meniscal and fabella bone volume, subchondral bone thickness and density and cartilage degradation. In contrast, dampened EGFR signalling had little effect on signs of PTOA in male mice, and in some instances (meniscal bone volume and subchondral bone thickness), it actually made them worse. Zhang et al. (2014) showed that the down-regulation of EGFR signalling via genetic modification or administration of gefitinib enhanced cartilage destruction post DMM surgery in male mice [187]. This study, together with our results suggest that EGFR signalling plays a sex dependent role in the development of PTOA in mice, protecting against PTOA in females but having no effect or worsening the signs of PTOA in males. Interestingly, estrogen receptors are known to be involved in ligand-independent activation of EGFR on tyrosine 845 (Y845) via G- protein-coupled receptor and Src kinase in human breast cancer cells [222, 223] and it has been shown that erlotinib significantly reduces both ligand-dependent and independent EGFR phosphorylation [224]. Although both male and female growth plate chondrocytes express estrogen receptors, the mrna and protein levels of estrogen receptors are higher in chondrocytes from female compared to male mice [225, 226]. Based on this evidence, we hypothesise that the 61

79 sex dependent effect of erlotinib treatment on PTOA seen in our study is due to higher ligandindependent activation of EGFR by estrogen receptors in female compared to male mice. Further studies are required however to test this hypothesis in mouse chondrocytes and osteocytes. In addition to dampening the signs of PTOA, erlotinib treatment also lessened changes in numerous trabecular, fabella, and subchondral bone parameters that occurred independently of DMM surgery. This result was also sex dependent, evident in female but not male mice, and suggests that EGFR signalling can also influence bone remodelling processes that occur due to normal skeletal development. In support of this finding, it was previously reported that administration of gefitinib into 1-month-old rats produced significant defects in endochondral ossification, including thickening of epiphyseal growth plate and accumulation of hypertrophic chondrocytes, which highlights the critical role of EGFR signalling in the remodelling of growth plate cartilage during skeletal development [227]. In this study we have performed DMM surgery on integrin α1-null mice for the first time, confirming success by observing the dislocation of the medial meniscus by microct analysis. The effects of surgery were observed as early as 4 weeks PS in both soft (i.e. cartilage degeneration and synovitis) and calcified (i.e. higher bone volume and density of menisci and fabella, and thickening of subchondral bone) tissues of DMM compared to sham and contralateral joints. As expected, these signs were primarily found in the medial compartment of the knee and the changes conform to the characteristic signs of OA observed after DMM surgery [192, 197, 228]. The delay of cartilage damage in females compared to males PS agrees with others who have reported that male hormones aggravate and female hormones protect against the effects of DMM surgery up to 8 weeks PS [158]. Despite this initial protection, our results suggest that by 12 weeks PS females experience a similar severity of cartilage damage as their 62

80 male counterparts. Interestingly, twice as many females compared to males developed a calcified MCL, and the increase in meniscal bone volume seen post DMM was larger in female compared to male mice. Together, these results highlight the importance of considering sex dependent responses and changes in both soft and bony tissues of the knee when using the DMM model of PTOA in mice. In conclusion, we have shown that integrin α1β1 protects against PTOA-induced cartilage degradation up to 8 weeks post DMM surgery partially via the dampening of EGFR signalling, but only in female and not male mice. Furthermore, we showed that the dampening of EGFR signalling plays a sex dependent role in the development of PTOA in cartilage, bone and meniscal tissues in mice, protecting against PTOA in females but having no effect or worsening the signs of PTOA in males. Future studies will examine the influence of interplay between the estrogen receptor and EGFR in the sex dependent role of EGFR signalling in the responses of knee tissues to PTOA. 2.5 Funding This work was supported by CIHR MOP (AC), CIHR Studentship (SS), Alberta OA Team Studentship (SS), NSERC CREATE Incentive Award (SS), University of Calgary Faculty of Kinesiology (SS), the Veterans Affairs Merit Reviews 1I01BX (AP), and the National Institutes of Health grants DK (AP). 2.6 Acknowledgements The authors would like to thank Genentech, Inc. and Astellas Pharma, Inc. for provision of Erlotinib hydrochloride, Carin Pihl and Dawn Martin for expert assistant in all animal procedures, 63

81 Britta Jorgenson for microct expertise, Hakan Kadir and Charlie Shin for gavage assistance, Erica Floreani, Lisa Milo and Hakan Kadir for microct and histology assistance, and Dilene Mugenzi for histology assistance. 64

82 Chapter Three: Behavioural Testing 3.1 Introduction Behavioural tests are often used in the literature to assess general well-being and pain levels in mice [229]. For example, mechanical hypersensitivity (allodynia) of mouse paws, measured using von Frey hairs, can be observed in limbs with induced knee OA [230, 231]. In addition, mouse gait can be analyzed by taking paw prints of unprovoked motion in a straight line [232]. Mice with OA-like signs at the knee, such as in the case of mice deficient in type IX collagen, were shown to have shorter stride lengths compared to wildtype (WT) mice, suggesting that gait can measure functional consequences of OA [232]. By pairing behavioural tests in mice with the assessment of bony and soft tissue changes in the knee, it may be possible to characterize the severity of pain relative to the morphological changes in the knee over time. Such data would provide valuable, more clinically relevant insight into the initiation and progression of PTOA. This triangular research design is a novel approach to investigate the effect of integrin α1β1 on PTOA progression. The goal of this study was to examine the role of integrin α1β1 on the progression of PTOA. We hypothesized that the behavioural effects of PTOA, characterized by altered amount and type of activity, gait patterns, grip strength and pain, would be exacerbated in α1-null mice at every time point compared to WT mice. Furthermore, we hypothesized that treatment with the EGFR inhibitor erlotinib would dampen the effects of integrin α1β1 on these behavioural signs of PTOA. 65

83 3.2 Materials and Methods Twenty-four male mice, with equal numbers of surgery type (DMM/sham), genotype (α1- null/wt) and treatment type (erlotinib/vehicle), were randomly chosen from the 12 weeks PS group (Appendix A). Due to the limited number of mice logistically possible to include in the behavioural experiments, the sex variable had to be removed to ensure a sample size of three mice per group. Male mice were chosen to undergo behavioural testing as males were previously shown to develop more severe signs of PTOA compared to female mice [158]. A Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS; Metris, Hoofddorp, Netherlands) was used to monitor the behaviour of these mice for 23 hours (12 hours of night and 11 hours of day) at 4, 8, and 12 weeks PS. The LABORAS system has been validated to record and classify mouse behaviour into five categories: resting, grooming, eating, locomotion, and climbing according to the frequency of vibrations of a sensitive force platform [233]. Analyses were conducted on the LABORAS data across the entire 23 hours of collection as well as just during their most active time (8pm 12am) which was defined as the least amount of time spent immobile across all mice. Mouse strength, sensitivity, and gait were assessed at 4, 8, and 12 weeks PS by grip strength, von Frey hair, and stride length testing, respectively [ ]. For grip strength testing, mice were placed in a scruff hold and held such that one of their hindpaws grasped the grip strength meter (Columbus Instruments, Columbus, OH). The mouse was then pulled backwards away from the machine and the maximum force applied by the mouse paw on the machine during that motion was recorded. Measurements were made using both surgical and contralateral hindlimbs, and three trials were performed per mouse. 66

84 The mechanical sensitivity of the plantar surface of the mouse foot was measured using a von Frey hair testing procedure detailed by Fuchs et al. (1999) [234]. Essentially, von Frey hairs increase in thickness and thus stiffness and are calibrated to apply a specific millinewton value of force to the mouse foot. Mice were acclimatized to a wire-bottomed cage and then von Frey hairs were applied in an order of ascending force magnitude to the center of the plantar surface of a hind paw. The withdrawal response of the mouse leg associated with the stimulus was recorded. This sequence was repeated three times on both left and right hindpaws with a 30 minute rest period between each trial. The average frequency of paw withdrawal for each level of von Frey hair stimulus was recorded. Gait was assessed via stride length test. All four paws of each mouse were painted with food colouring of contrasting colour and the mouse then placed at the open end of a custom-built acrylic gait track attached to a dark enclosed cage at the other end. A layer of plain white paper lined the bottom of the track, and upon release, the mouse ran to the cage and left paw prints on the paper. Three strides in the middle of the track were used per trial to obtain average stride length for both sides and stance width. A cool down time of 30 minutes to an hour was provided between tests to minimize stress Statistical Analysis A repeated measure MANOVA was used with three between-subject factors (genotype (α1-null or WT), surgery (DMM or sham), and treatment (erlotinib or vehicle)) and one (time PS) or two (time PS and leg) within-subject factor. A Bonferroni post hoc test was performed to determine significance at p

85 3.3 Results Contrary to our hypothesis, DMM surgery, genotype and erlotinib treatment did not significantly influence any of the behavioural parameters measured. Erlotinib decreased the time spent immobile and increased the time spent climbing (Figure 3.1) but these changes did not reach statistical significance. Figure 3.1 Time spent immobile (A) and climbing (B) as a function of treatment (vehicle or erlotinib) over 23 hours of test duration. Data points represent mean of n = 3 trials for N = 12 mice ± 95% confidence interval. Note the trend (though statistically insignificant) of less time spent immobile (A) and more time spent climbing (B) in erlotinib compared to vehicle treated mice. Furthermore these changes were small (immobile 41 mins and climbing mins) relative to the 23 hours of recording time (1380 mins) and thus the clinical relevance of these changes may be limited. Finally, erlotinib treatment restored the decreased locomotor activity in mice following both DMM and sham surgery during the 4 hours between 8pm and midnight when the mice were most active (Figure 3.2). These observations however were not statistically significant. 68

86 Figure 3.2 Time spent in locomotion at 4, 8, and 12 weeks post destabilisation of medial meniscus (DMM) or sham surgery as a function of treatment (erlotinib or vehicle) during their most active time (8pm 12am). Data points represent mean (N = 6 mice) ± 95% confidence interval. Note the trend (though statistically insignificant) of less time spent in locomotion for vehicle treated joints at 8 and 12 weeks PS compared to 4 weeks PS. 3.4 Discussion DMM surgery, genotype and erlotinib treatment did not significantly influence any of the behavioural parameters measured. However, interesting but statistically insignificant trends were found in mouse activity measured via LABORAS. Based on their unpublished data, Fang et al. (2014) suggest that the development of OA in mice primarily leads to alteration in locomotor activity levels rather than gait kinematics, which could explain why trends were only observed in LABORAS and not stride length measurements in the current study [229]. Provided the two independent studies have published significant alterations in activity levels observed as early as 8 weeks post DMM surgery with a minimum sample size of 10 in each group [196, 235], it is possible that our study lacked in power to show the behavioural alterations with 12 or 6 mice respectively in each group shown above. 69

87 The optimum sample size for a given statistical significance and power can be calculated using the following equation [236]: n = 4σ2 (Z a + Z 1 β ) 2 2 Figure 3.3 Conservative sample size determination for repeated measures analysis where Zα is a standard score of chosen level of significance and Z1-β is a standard score of chosen power. The symbol σ is the standard deviation and is the estimated effect size. We can use the accepted values for level of significance of 5% (two sided) and power of 80% with corresponding standard scores of 1.96 and , respectively and the data above to obtain values for standard deviation and estimated effect size (σ = , = for immobile time, σ = , = for climbing time and σ = , = for locomotor time). Using these values, the sample size required for the effects to reach significance are 26, 24, and 36 respectively. Unfortunately, the logistics of daily gavage and availability of LABORAS machines limited the number of measurements that could be performed in a week, and thus we were only able to use three mice in each genotype, surgery, and drug group. An improved study design to resolve these limitations would be to collect data in multiple sets over a longer period of time. For example, testing three mice per group for three months and repeating the measurements twice, each with another set of mice per group, would yield a total of nine mice per group over the duration of nine months. However, longer duration study designs have their own limitations, such as additional sources of variability between earlier and later tests, researcher fatigue and higher cost. 70

88 Chapter Four: Discussion 4.1 Hypothesis and Specific Aims Based on the literature and preliminary data presented in Chapter 1, we hypothesized that: 1. The effect of PTOA, characterized by loss of cartilage, growth of osteophytes, and decreased activity levels, would be exacerbated in integrin α1-null mice at every time point compared to WT mice. 2. The protective mechanism of integrin α1β1 against PTOA involves the dampening of EGFR activation. To test these hypotheses, we performed DMM surgery on integrin α1-null and WT mice and monitored the progression of PTOA over 12 weeks using micro-computed tomography, histology, and behavioural testing. Furthermore, we administered erlotinib to test whether EGFR signalling is one mechanism by which integrin α1β1 protects the knee from PTOA. As such, the specific aims of this study were: 1. To quantify changes in the calcified tissues of the knee using micro-computed tomography, and changes in articular cartilage and synovium using histological methods, in integrin α1-null and WT mice at 2, 4, 8 and 12 weeks post DMM surgery. 2. To quantify changes in withdrawal threshold, grip strength, stride length, and activity levels in male integrin α1-null and WT mice at 4, 8 and 12 weeks post DMM surgery. 3. To administer erlotinib to an additional group of integrin α1-null and WT mice from the day after DMM surgery until sacrifice at 12weeks post-surgery to elucidate the role of EGFR signalling as a mechanism by which integrin α1β1 protects against PTOA. 71

89 4.2 Summary of Key Findings Integrin α1β1 Offers Less Protection Against PTOA Than Spontaneous OA For the first time, we elucidated the role of integrin α1β1 in the progression of PTOA, enabling us to compare with its known protective role against spontaneous OA [157, 161]. We found that though integrin α1β1 protects against signs of both spontaneous and post-traumatic OA, the effects were smaller and tissue dependent in PTOA in contrast to spontaneous OA. This may suggest that the mechanism by which integrin α1β1 protects against OA, and the downstream pathways involved, may be different between various subtypes of OA as well as different tissues of the joint. Alternatively, these differences may be explained by contrasts between these subtypes of OA, such as the involvement of knee trauma and/or mechanical instability, and the timeframe of disease progression (weeks vs. months) [157, 161, 192]. A serious trauma to the knee, such as anterior cruciate ligament tear or DMM surgery, exposes the joint to blood which is not normally present within the knee capsule. Long-term (i.e. hemophilia) as well as short-term exposure to intra-articular bleeding has been shown to induce both OA-like cartilage destruction and inflammatory processes associated with rheumatoid arthritis [195, 237, 238]. These rapid inflammatory responses to bleeding in PTOA which are presumably diminished in spontaneous OA may be larger than and overwhelm the protection offered by integrin α1β1. Shorter timeframe of disease progression in PTOA is another factor that may influence the role of integrin α1β1. Although we are unable to pinpoint the exact timeframe of spontaneous OA progression in integrin α1-null mice due to the lack of definite starting point, according to our previous work and other literature, it occurs between 6 to 9 months post skeletal maturity (3 72

90 months of age) [157, 161]. On the other hand, signs of PTOA in α1-null mice were observed as early as 4 weeks post DMM surgery in skeletally mature mice. This faster onset of joint damage in PTOA compared to spontaneous OA may have decreased the time that integrin α1β1 and its downstream pathways have to influence disease progression, thus leading to reduced protection Dampening Epidermal Growth Factor Receptor Signalling Protects Against PTOA in a Sex dependent Manner The effects of the EGFR inhibiting drug erlotinib on signs of PTOA in α1-null mice were studied for the first time. As expected, erlotinib treatment diminished signs of PTOA in both soft and bony tissues of the knee, but the mitigating effects of the drug were primarily seen in female and not male mice. Furthermore, though the effects of erlotinib were observed in both α1-null and WT mice, it protected against cartilage damage to a greater extent in α1-null compared to WT mice. This result suggests the potential involvement of EGFR signalling as one mechanism by which integrin α1β1 protects against PTOA, however the tissue and sex dependent nature of these results require further investigation. The sex dependent response to erlotinib may be explained by the varying level of interplay between estrogen receptors and ligand-independent phosphorylation of EGFR as discussed in Chapter 2. This could be examined by performing DMM surgery on ovariectomized female mice and treating them with erlotinib to see whether the lack of estrogen has any influence on the effect of erlotinib on PTOA progression. We would expect the protective effect of erlotinib to be less in ovariectomized mice compared to control, as measured by histology and microct. Understanding the mechanism of the sex dependent response to erlotinib would allow us to select the most effective population of mice to use for future studies into the use of erlotinib to treat OA. 73

91 In order to further understand the tissue dependent response to erlotinib ex vivo and in vitro experiments could be performed. Previously, calcium transient responses of chondrocytes on intact femur to TGF-β and IL-1 have been used to measure differences in chondrocyte sensitivity to these ligands in α1-null and WT mice [11]. In addition, a downstream product of EGFR activation are reactive oxygen species (ROS), which could be detected in a similar manner to calcium transients ex vivo under confocal microscopy using dihydroethidium [239]. Such ex vivo studies comparing α1-null and WT chondrocytes could be complimented by in vitro studies of both chondrocytes and osteocytes of α1-null and WT mice. For example, these cells could be incubated with dihydroethidium and then exposed to media containing TGF-α, an EGFR specific substrate, to see if the level of EGFR signalling is different in the two types of cells. Furthermore the cells could be additionally exposed to erlotinib to confirm that EGFR signalling is inhibited by the drug and to compare the extent of EGFR inhibition in the two types of cells. We hypothesize that EGFR signalling is greater in osteocytes compared to chondrocytes and/or that erlotinib inhibits EGFR signalling to a greater extent in osteocytes compared to chondrocytes. Furthermore we hypothesise that EGFR signalling would be greater in α1-null compared to WT cells. Such experiments would help to explain the enhanced protective effect of erlotinib in bone compared to cartilage tissue, and in the cartilage of α1-null compared to WT mice Female Mice are Protected from Post-traumatic Osteoarthritis up to 8 Weeks Postsurgery Another important finding of this study is the sex dependent response to DMM surgery. Previously it was noted that estrogen protects and testosterone aggravates the signs of PTOA, which explains the reason why female mice are protected against the signs of PTOA up to 8 74

92 weeks post-surgery [158]. Our results confirm this protective effect observed in female mice, however, we also show that by 12 weeks post-surgery, females experience a similar severity of cartilage damage as their male counterparts. The sudden appearance of signs of PTOA in females at 12 weeks PS is not clearly understood. It is unknown whether estrogen levels fluctuate after a prolonged exposure to joint injury or if other pathways are activated after 8 weeks that overwhelm the protection offered by estrogen in female mice. Monitoring of estrogen levels and providing 17-β estradiol supplements following DMM surgery in female mice may provide the basis for further studies looking at the effect of fluctuating estrogen levels on the risks of developing signs of PTOA following injury. Together, these results highlight the importance of considering sex dependent responses and changes in both soft and bony tissues of the knee when using the DMM model of PTOA in mice. 4.3 Strengths and Limitations Animal Model Animal models are powerful tools that allow us to study the pathogenesis as well as pharmaceutical interventions of many diseases in ways that are not be possible using human subjects. The instability induced animal models of osteoarthritis closely resemble the characteristics of human arthritis that develop after a traumatic injury [240]. Mice, rabbits, cats, dogs, and even horses are used to study PTOA and each model has its own advantages and disadvantages that make it ideal for answering certain hypotheses [241]. For example, larger animal models may better represent the anatomical size and features of a human knee, but the cost of using them may be inhibitive in large scale studies. On the other hand, mice are relatively 75

93 low cost and easier to maintain, but their thin cartilage makes it a difficult model to study cartilage morphology. The unique advantage of using mice models is that the mouse genome has been sequenced, which allows the production of mice with modified genes. This ability to genetically modify the expression of a protein of interest offers researchers a way to characterise the phenotype of an organism with modified expression of the protein in vivo, and thus provides an insight into the role of the protein related to the phenotype observed. In this study, we used α1-null mice to elucidate the role of integrin α1β1 in the development of PTOA. One limitation of our use of mice carrying a global knockout of the integrin α1 subunit is that the changes in cartilage may not necessarily be a result of chondrocytes lacking in integrin α1β1, but rather may be due to the changes in other tissues that affect the morphology of cartilage. Furthermore, although no obvious phenotypic differences have been measured in α1- null and WT mice, there may be other unknown signalling pathways that compensate for the lack of integrin α1β1 and influence our experiments. It is important to note however, that no compensatory up regulation of other integrins in response to the lack of integrin α1β1 has been measured in α1-null mice [215]. An additional limitation of our animal model is that studying the effects of the loss of integrin α1β1 may not directly translate to the effect of increasing its expression as seen in early OA [157, 203]. For example, observing earlier onset of PTOA in α1- null mice does not necessarily mean that the upregulated expression of integrin α1β1 in WT mice would protect against PTOA. Despite these limitations, as far as we are aware, there is no other way of directly influencing the expression and/or signalling of integrin α1β1 in vivo, and genetically altered mice still provide valuable insights that form the basis of future studies related to human OA. 76

94 4.3.2 Erlotinib Administration EGFR activity can be altered genetically or pharmaceutically using drugs such as erlotinib or gefitinib [187, 189]. An alternative approach would be to create mice lacking both the integrin α1 subunit and EGFR however this would require multiple generations of crossbreeding. Consequently, the use of erlotinib was the most ideal to study the relationship between integrin α1β1 and EGFR in α1-null mice. Erlotinib and gefitinb are approved by the Food and Drug Administration for treating advanced non-small cell lung cancer, hence the safety of these drugs has been verified in many animal models as well as in humans [190]. At the planning stages of our study, erlotinib had been used to inhibit EGFR and effectively treat arthritis (albeit rheumatoid arthritis) in mice, and thus erlotinib was the drug used in our study [189]. One limitation of the administration of erlotinib is that the orally ingested drug can be taken up by any tissues of the body. For our study, it would be helpful to know how much erlotinib was been taken up by knee tissues. Furthermore, we used a constant dosage (50 mg of drug/kg of mouse/day) for all mice based on their average mass, which would have led to small variations in the dosage received per mouse. As female mice in our study weighed approximately 5g less on average than males, females would have received a slightly higher dosage compared to male counterparts. However, it is important to note that due to the practical limitations of oral gavage and the limited solubility of erlotinib, the errors involved in the administration of erlotinib in our study were likely larger than the error due to the difference in mass. For example, although we thoroughly mixed the solution at every instance of gavage, erlotinib is not very soluble and thus some mice may have received more than others. Furthermore, occasionally some of the drug would remain in their mouth despite proper gavaging techniques, making it difficult to control how much they ingest. 77

95 The bioavailability of the drug is also important to consider. When taken on an empty stomach, erlotinib has an oral bioavailability of 60% [242]. However, this increases dramatically up to 100% when taken with food [242]. Although mice were treated with erlotinib during the inactive part of their day (noon), they may have had varying levels of food in their stomach which would have affected the bioavailability of the drug. However, the amount of food each mouse ingested was not controlled in our study due to housing them in large groups. Finally regarding our use of erlotinib, it is possible that after 24 hours there was little or no drug left in the mouse. It is known that after approximately 10 hours of 5mg/kg erlotinib oral administration, the bioavailability of the drug is close to zero per cent in plasma concentrations of mice [243]. Despite our use of ten times as much erlotinib (50mg/kg), it is possible that the effect of the drug would wear off during the 24 hour period between gavages. Increasing the frequency of gavage was considered but deemed impractical for a study lasting three months and using 80 mice (40 erlotinib and 40 vehicle) Skeletal Maturity DMM surgery has most often been performed at 12 weeks of age [187, 228, 244] in mice, but in some instances it was performed as late as 20 weeks of age [245] or as early as 10 weeks of age [198]. A study analysing the growth of C57BL/6 mice concluded that female mice are not skeletally mature until 20 weeks of age, however a more recent study that included both sexes reported that the rate of growth of C57BL/6 mice starts to slow at 12 weeks of age and they reach skeletal maturity at 4 months old [246]. Therefore, assuming that the growth rate of BALB/c mice is comparable, we performed DMM surgery at 12 weeks of age. 78

96 Interestingly, we observed a gradual increase of fabella volume and trabecular density and thickness regardless of surgery over the 12 weeks of our study suggesting that the bones were still remodelling beyond 12 weeks of age. This bone growth and tissue remodelling occurring with growth may have dampened the effects of surgical intervention. Though it was a confounding variable, this led to the discovery that erlotinib can also influence bone remodelling processes that occur due to normal skeletal development, in addition to PTOA Incorporation of Behavioural Tests Pain and disability are key clinical features of OA that lead to its diagnosis. Although the consensus is that OA pain and functional impairment follow significant joint damage, the relationship between knee OA pathology of different tissues and observed pain levels in humans remain unclear [ ]. Several outcome measurements have previously been used to measure pain and disability in animal models, including recording spontaneous activity levels, gait, grip strength, and mechanical allodynia [191]. Interestingly, the majority of OA pain research in animals uses chemically induced models of OA, such as intra-articular injection of monoiodoacetate, that have pathology that is atypical of human OA [191]. By monitoring the changes in mouse behaviour in parallel to the morphology of soft and bony tissues of the knee during PTOA development, we aimed to characterise the time of onset as well as the severity of pain relative to the morphological changes that mimic human OA. Observing reduced OA pain in erlotinib compared to vehicle treated mice and/or WT versus α1-null mice would provide the basis for further studies testing the effectiveness of pharmaceutically targeting EGFR or integrin α1β1 to arrest the progression of PTOA and its associated pain. 79

97 We performed various behavioural tests on a subset of mice including stride length testing, muscle strength, and activity levels, but found no significance effects related to surgery, erltoinib treatment or genotype. One reason for this lack of behavioural difference may be that there was no behavioural alteration due to moderate or low levels of pain in mice with PTOA. Selective pressure has led to the evolution of rodents, such as mice, that do not appear sick or lame in order to avoid predators, which present difficulties when measuring pain and disability in mice [250]. As such, despite the significant morphological changes associated with PTOA, the mice in this study may not have displayed any abnormal behaviour. Alternatively, since other studies [196, 235] have shown significant behavioural changes post DMM surgery in mice, it may be possible that our study did not have enough power to measure significant behavioural alterations. Due to the logistical limitations imposed by the availability of LABORAS machines and the requirement of daily gavage, we were limited to including three mice in each genotype, surgery, and drug group. Another potential reason for this lack of significance may be that the effect of pain post DMM surgery on behaviour was outweighed by other factors such as short term stress. As the consistency of behavioural tests depends heavily on the cooperativeness of mice, stress would have been another factor that affected their behaviour. For example, the withdrawal response to von Frey hair stimulation may be difficult to classify if the mouse is hypersensitive due to stress. This may underestimate the significant behavioural differences between DMM and sham mice. Though we acclimatized the mice to the device prior to testing days to minimize stress, mice had to be physically transferred from housing (in the basement of the Health Sciences building) to the behavioural facility (on the first floor of the Health Sciences building) and back on every testing occasion which may have increased their stress. The order of tests as well as the time of testing 80

98 may have also influenced these results. We observed that mice would continuously groom immediately after stride length testing, thus we ensured that stride length test was performed last and although cool down time of 30 minutes to an hour was provided between tests, it may not have been enough to reduce stress to negligible levels. Furthermore, as the tests were performed during their inactive time of day (except LABORAS which was measured for 23 hours), the behavioural patterns measured may not represent their normal daytime behaviour. 4.4 Future Studies The conclusions we have drawn about the protective effects of erlotinib on the progression of PTOA are based on the assumption that erlotinib was taken up by the tissues of the knee. Therefore, it would be important to verify the level of EGFR signalling in knee tissues in the mice of our study. Immunohistochemistry of phosphorylated EGFR is currently being performed on the same mouse joints used in this study to test the effectiveness of the erlotinib treatment in dampening EGFR signalling in chondrocytes and osteocytes. Metabolomics analysis of serum collected from the mice used in this thesis is also being performed to compare the level of different proteins across surgical intervention, genotype, and sex. Preliminary results indicate similar trends to what we observed in this thesis such as sex, drug, and surgery effects of protein levels in serum, and this would help us to identify metabolic alterations that help explain the differences observed. Furthermore, biomarkers of PTOA progression may be identified which would provide the basis for preclinical studies testing the use of serum metabolites in the early diagnosis of human OA [251]. Unfortunately, due to the logistical reasons described earlier, we were limited in the number of mice we could include in the behavioural testing portion of our work. Based on a 81

99 different study that reported that female mice were protected against induced PTOA up to 8 weeks PS, we chose to only include males in the behavioural testing. However, this thesis showed that although female mice are protected against signs of OA up to 8 weeks PS, they develop significant signs of OA that are equivalent in the magnitude of damage to age-matched male mice at 12 weeks post-surgery. Given this, since the majority of the erlotinib effects were seen in female mice, it would be interesting to know whether there would be any significant behavioural alterations that would be detectable in female mice. We could replicate the behavioural part of this study with a higher sample size of female mice to demonstrate the clinical relevance of the erlotinib effects in the progression of PTOA in female mice. Lastly, rather than a global knockout of the integrin α1 subunit, chondrocyte-specific knockout could be produced using a cre-lox mouse such as tamoxifen regulated collagen II Cre mice [252]. This would allow us to examine the role of integrin α1β1 in chondrocytes specifically and how the alterations in cartilage affect the rest of the joint tissue (i.e. bone and synovium). Similarly, osteocyte-targeted α1-null mice can also be produced to study the effects of integrin α1β1 in bony changes during OA progression [253]. 4.5 Conclusion In conclusion, we have shown that integrin α1β1 protects against PTOA-induced cartilage degradation up to 8 weeks post DMM surgery partially via the dampening of EGFR signalling, but only in female and not male mice. Furthermore, we showed that the dampening of EGFR signalling has a sex dependent effect on the development of PTOA in cartilage, bone and meniscal tissues in mice, protecting against PTOA in females but having no effect or worsening 82

100 the signs of PTOA in males. Taken together, these findings highlight integrin α1β1 and/or EGFR signalling pathways as potential pharmaceutical targets for alleviating the signs of knee PTOA. Due to the lack of symptoms in its initial stages, the detection of spontaneous osteoarthritis often occurs years after joint damage has begun. This makes it difficult to implement early interventions that might prevent or slow down the progression of the disease. In contrast, the clearly defined initiation of PTOA at the time of injury enables these high risk individuals to be identified and may allow for early intervention before any signs of osteoarthritis arise. This thesis provides a first step in investigating integrin α1β1 and/or EGFR as potential pharmaceutical targets that may delay or prevent PTOA progression, and thus improve the quality of life of injured individuals and reducing healthcare costs related to joint rehabilitation, surgical intervention and pain management. 83

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119 102 Appendix A: DMM EXPERIMENT TIMELINE 100 WT mice 100 KO mice (12 Week Old Mice) SURGERY 0 Weeks 2 Weeks 4 Weeks 8 Weeks 12 Weeks 176 Mice Half of each sex * 10* 10* 10* 10* 10* 10* 10* 10* 10* 10* 10* 7* 7* 7* 7* 7* 7* 7* 7* Behavioural Testing (24 Mice - Male only) (12 Week Old Mice) SURGERY 0 Weeks 4 Weeks 8 Weeks 12 Weeks LEGEND WT Mice KO α1 Mice SHAM Surgery DMM Surgery Erlotinib treated throughout 12 weeks post surgery. All other mice in 12 weeks group will be administered with a control * Indicates Sacrifice at Time Point DMM destabilisation of medial meniscus 3* 3* 3* 3* 3* 3* 3* 3*

120 Appendix B: TARCEVA PREPARATION PROTOCOL Written by Lisa Milo Dosage 50 mg/kg/mouse/day Mouse = 30g 0.03 kg 50 mg/kg/mouse/day x 0.03 kg = 1.5 mg/mouse/day In 0.1 ml because that s what a mouse s stomach can hold 1 Tarceva Tablet = 100 mg Erlotinib Concentration : 1.5 mg 0.1 ml 100 mg V = 15 mg/ml = 15 mg/ml Dissolve each tablet in ml of 0.5% (w/v) of (hydroxypropyl) methyl cellulose and 0.1 % (v/v) of TWEEN 80 in distilled water 100 mg 15 mg/ml = V ml = V *Store at -20 and defrost and mix well before administering Materials: Mortar and Pestle Weighing Paper Erlenmeyer Flask (125 ml) Distilled H 2 O 1000 μl Pipette 1000 μl Pipette Tips (VWR, ) 1 Falcon Tube (15 ml) per tablet (15 ml, VWR, ) Micro-centrifuge tubes (1.5 ml. VWR, ) TWEEN 80 (Sigma-Aldrich, P1754) Needle Tip (20 gauge) Tube boxes Stir bar (Hydroxypropyl) methyl cellulose (HPMC) (Sigma-Aldrich, H G) Tarceva (tablets) (100mg erlotinib hydrochloride/tablet, Roche, ) HMPC Solution [500 mg/100 ml HPMC (Sigma-Aldrich, H G) and 100 mg/100 ml TWEEN 80 (Sigma-Aldrich, P1754)] Preparation of HPMC Solution a. Fold weight paper in half and place on scale 103

121 b. Zero the scale c. Place ~ 500 mg (0.5 g) of HPMC on the weighing paper (approximately the size of a quarter) d. Transfer into Erlenmeyer Flask (125 ml) e. Add 100 ml of distilled H 2 O into the Erlenmeyer flask f. Put in 1 magnetic stirring bar g. Cover Erlenmeyer flask with tinfoil h. Place Erlenmeyer flask on stirrer i. Leave overnight or until solution is completely clear with no slimy bits j. Once solution is clear add 100 mg (2-3 drops) of TWEEN 80 using a 1000 μl pipette k. Stir till solution is clear l. Add to stock solution of HPMC *Add TWEEN 80 after stirring because it is a detergent and creates bubbles Tarceva Preparation a. Label micro-centrifuge tubes Pink, Orange, Yellow are HPMC and Purple, Blue, Green are Tarceva b. If using Tarceva tablets, place 1 Tarceva tablet in a dry mortar and grind tablet using pestle until it is a fine white powder. Alternatively weigh 100 mg of Erlotinib (approximately loonie sized) provided by Genentech on a piece of weigh paper and shake the powder into the mortar. c. Scrape powder off of end of pestle using a pipette tip or spatula if using Erlotinib from Genentech tap the weigh paper over the mortar to get all of the powder off. d. Using the 5 ml pipette add 3 ml of HPMC solution to powder e. Mix liquid and powder using a 1000 μl pipette set to 500 μl swirling the tip in the liquid and pipetting up and down f. Clean the pipette with the needle (20 gauge) if clogged g. Mix until no visible bits of tablet are present h. Transfer solution into Flacon Tube (15 ml) i. Using 5 ml pipette add another 3 ml of HPMC solution to mortar j. Move liquid around mortar using a 1000 μl pipette tip k. Transfer liquid to falcon tube l. Add 700 μl HPMC solution to falcon tube using a 1000 μl pipette making 6.7 ml of solution m. Wipe out and dry the mortar out with paper towel in between tablets n. Vortex the tube periodically until the liquid does not appear to separate (2-3 hours). If using Erlotinib provided by Genentech tubes should be placed on rotator and shaken constantly for 8 hours to mix into solution. They should also be taken off the rotator and vortexed periodically. o. Once liquid no longer separates, transfer 1 ml of Tarceva into pre-labeled microcentrifuge tubes p. Spin falcon tubes in the centrifuge for ~1 minute at 1400 RPM and at room temperature (~20 ) q. Transfer any remaining Tarceva solution into pre-labeled micro-centrifuge tubes 104

122 r. Transfer 1 ml of HMPC solution to pre-labeled micro-centrifuge tubes s. Place all the tubes into a box t. Place box in ~20 u. Tablets will stay in solution but Erlotinib from Genentech will drop out even if frozen *DON T use pipettes with filter tips 105

123 Appendix C: MICE SACRIFICE PROTOCOL Written by SungYong (Kevin) Shin, edited by Lisa Milo *The following protocol includes blood, hindlimbs, and body collection. Materials list (bring extras) 1 Needle (25 gauge) per mice Tube Boxes (-80 C) 2 Micro- Centrifuge Tubes (blood) per mice (1.5 ml, Fisher, ) 2 Cryo tube (leg) per mice (2mL, Greiner Bio-One, ) 1 Syringe (3mL) per mice 2 Scalpel blades per mice PBS ph 7.4 (GIBCO ). A spray bottle of 70% alcohol 1 Zip lock bag (small) per mice Blue 'diaper' pads 1 Zip lock bag (big) per surgery date Jirik mice ID chart Large beaker for weighing mice Tools list 2 Scalpel Handles (1 for sac ing and another for dissection) 1 Tissue Scissors 1 forceps with teeth Surgical cutting board 1. The sacrifice and blood collection a. Get a bucket with ice ready. b. Label Cryo tubes for blood for each mouse. (ID, Sex) c. Label ziplock bags with each mouse ID. (ID, Sex) d. Get needle and syringe ready. Take cap off and pump the syringe. e. Weigh mouse using large beaker and scale f. Put mouse under using CO 2 Chamber. Put the lid on as mouse sometimes jumps. Wait until they lie down on one side and count 3-5 breaths. Alternatively, place a few pieces of dry ice inside a 500mL beaker placed inside a larger beaker or box partially covered. Pour hot water onto the dry ice to create vapours that fill the box. Holding a mouse by the tail submerge them in the vapours as low down in the box as possible until they relax. g. Quickly, take mouse out and spread out the two forelimbs using left hand. 106

124 h. Collect whole blood (~0.5-1 ml total) via cardiac puncture into pre-labelled cryo tube. i. Ensure mouse is dead by checking heartbeat. If mouse is not dead place gently in the corner of the CO2 chamber. j. Put dead mouse in the pre-labelled ziplock bag and place the bag on ice k. Let the blood clot on ice for at least 20 minutes. l. Spin the tubes for 20 minutes at 5000 rpm (2000 G s) at room temperature (~20 ). m. Using a 200μl pipette collect serum from the top of the blood and place in a separate tube n. Place in -80ºC 2. Removal of hindlimbs a. Label cryo tubes for both left and right leg of each mouse and fill half way with PBS, ph 7.4 b. Spray mouse legs with PBS, ph 7.4 c. Remove the skin around leg by tenting the skin and making an incision that allows the skin to be pulled off of the muscles with the forceps, leaving muscles intact (Figure C.1). d. Once the leg has been exposed, place the leg so it s flat on the table (Figure C.2). e. Cut the femur half way up (Figure C.3). f. Place the paw of the limb flat on the table and cut near where the paw meets the tibia (Figure C.4). g. Remove the sock of fur/skin from around the ankle using forceps h. Put limb into pre-labelled cryo tube (Figure C.5). i. Place in tube box and place in -80ºC. 107

125 Figure C.1 After tenting and making the incision the skin is pulled off. Figure C.2 The limb has to lie flat on the table before it can be removed. Figure C.3 Limb is removed above the knee as close to the hip as possible. Figure C.4 Removal of the foot at the ankle. Figure C.5 Sample ready to be put in the freezer. 3. Body storage a. Wet enough gauze soaking wet with PBS, ph 7.4. b. Place mouse onto its back and fold tail to lie between front paws (Figure C.6). 108

126 c. Take one piece of gauze and wrap it around the mouse with fibres running vertically (Figure C.7). d. Take another piece of gauze and wrap it at 90 to the other piece (fibres running horizontally) (Figure C.8). e. Place it in a small zip lock bag and be sure to remove all the air.(figure C.9). Group mice by surgery date and place all the zip lock bags into a labelled (by surgery date) big zip lock bag. f. Place in -80ºC. Figure C.6 Folding the tail between the paws of the mouse. Figure C.7 Mouse wrapped in first piece of gauze (vertical fibers). Figure C.8 Mouse wrapped with second piece of gauze (horizontal fibers). Figure C.9 Mouse wrapped in gauze and placed in Ziploc bag with air removed. 109

127 Appendix D: HISTOLOGY AND MICROCT PROTOCOL Written by SungYong (Kevin) Shin Tissue Fixation 1. Take samples out of the -80 freezer and let them defrost. 2. Label 100ml bottles and Pink tissue cassettes with large holes (mesh is not good) according to the samples you have. 3. Fill bottles with 100ml Neutral buffered formalin/formaldehyde. 4. Once the samples are completely defrosted, place the samples in the cassettes and put 2 cassettes into each bottle. 5. Leave for at least 48 hours. There is no maximum time that the samples can remain in formalin. Total time: 2 days µct Scanning -Preparation 1. Make a sample holder with 4 slots out of foam. It should snugly fit into 30mm scanning cylinder. The height should be about 3/4 of the cylinder (75/3 = 25mm). 2. Use orange acrylic paint to mark the slots (The markings should be vertical). Top View Photos of the foam Figure D.1 Descriptive diagram and photos of the foam used. The numbers in top view represent the positions of each joint. µct Scanning Sample Preparation 1. Before scanning, scan blank to warm up the machine. Use the same control file that you will be using for the actual scan. 2. Make a table illustrating which sample goes into which slot. 3. Take samples out of cassettes, put them into corresponding slots. 110

128 4. Put the whole foam into the 30mm scanning cylinder (PEI: Poly-ether-Imid, Yellow), aligning slot #1 with the line of the cylinder (if the line where cylinder is joined together is not at the center of the metal holder, mark a short red line at the center of the holder and align slot #1 with the red line). You must use yellow cylinder as white cylinder is not as resistant to formalin. 5. Fill the cylinder with formalin. The samples should be completely covered in formalin a. Make sure that the samples are as straight as they can be. They all should be 90 degrees to the one next to it. Vertical! 6. Parafilm the cylinder top to prevent evaporation. 7. Cut extra parafilm hanging over the scanning region. 8. Scotch tape around the edges of the parafilm. Total preparation time: 1 hour µct Scanning with uct 35 Scanco Medical 1. Log in as ID: Invitro Pass: made2in2ch 2. Type into Operator : Bonelab 3. Click on the scanner button (It looks like the machine). 4. Create a new sample. 5. Naming convention: AC_A1KO_Sample1_Sample2_Sample3_Sample4 6. *No underscores, spaces in name. Use all CAPS. a. Ex. AC_A1KO_WT01_WT02_KO01_KO02 7. Press save. 8. Record the sample number in your log sheet. 9. File Exit 10. Choose the control file: Clark-Mouse knee joint (#50). 11. Click Scout View, change angle to 30 degrees (if the joints overlap on the screen). Then again click Scout View 12. Click Reference Line. Ideally, we want 2 full stacks which take around 76.4 minutes. 13. Move the top line to the highest point of patella. 14. If necessary, hold shift and drag mouse to extend scanning region. Extend by 1 full stack. 15. Low line should be just below the growth plate. 16. Click left mouse button to set the reference line. 17. Ok Scan Start Measurement 18. Record Measurement number. Total scanning time: 76.4 minutes, or around 85 minutes per scan including warm up. How to Batch Scan 1. Batch scan is useful when you have lots of samples and would like to save time preparing the samples. You may have a total of 8 samples in a foam holder, 4 on each plane. Make sure you leave enough room between the planes (~3cm). 2. Create both Sample names using the protocol above. 3. Find the reference line as indicated above. 4. Instead of starting measurement right away, click Add Scan 5. Under measurement sample, click other 111

129 6. Find the next 4 samples in the foam using the sample # recorded previously. 7. Add reference line, then add scan. 8. Exit. 9. Click Task List. There should be 2 measurements listed. Check. 10. Once checked, click Submit Batch Scans. Decalcification (All done in fume hood) 1. Trim muscles of all samples. 2. In the fume hood, pour out formalin into the formalin waste bucket. 3. Take samples out of cassettes and rinse them with deionized water. Keep cassettes in a bottle. 4. Label 50ml cylinder tubes with the sample names. 5. Put 1 sample in each tube and fill 50ml of fresh CalEX. 6. Leave it overnight. 7. Next day, label clean 15mL tubes with sample names. Shake 50mL cylinder tubes containing the sample. 8. Test if the samples are decalcified by taking 8ml of the CalEX solution from the sample tube and 2ml of 5% ammonium Oxalate (5g into 100ml of distilled water) into a 15mL tube. Mix well, keep it in a safe place. 9. Pour out the old CalEX from each of the sample tubes into waste, making sure the sample stays in the tubes, and pour fresh 50ml CalEX into the tubes. 10. Repeat with each sample. 11. After 2 hours (or overnight), check if there are any sediments on the bottom). If sediment is present, the samples are still decalcifying. 12. Repeat steps 5~9 until the test solution becomes crystal clear for 2 consecutive days. (Usually by the second day, crystal clear. Replace CalEx on Third day, then wait 3 more days to move on to A.T.M. On the sixth day, move on to A.T.M.). 13. Maximum number of days the samples may be in CalEX is 7 days. 14. Check if wax in the automated tissue processor is filled up to the line and clean. If not, fill. Total decalcification time: 6 days Automated tissue processor 1. Cut Femur and Tibia as close to the trimmed muscles. 2. Must have at least half of wax in the embedding machine wax container. If not, pour more (make sure wax is clean). 3. Place samples back into their cassettes. 4. Wash the samples with 1L room temperature tap water into 1L bottle for at least for 30 minutes. Keep the water running. To save water, run it for 10 minutes, replace with fresh water, and leave for 20 minutes. Wash until the ph reaches around Pour old chemicals out if previously used by someone else. Put fresh chemicals in according to the following table: 112

130 Table D.1 Table outlining the type of chemicals and time spent in each container of automated tissue processor. Container Chemical (2L, at least up to first line, Max second line) Length of time (hours) 1 Tap Water 1 2 Neutral Buffered formalin/formaldehyde % Alcohol Reagent alcohol and dh 2 O % Alcohol Reagent alcohol and dh 2 O % Alcohol Reagent alcohol and dh 2 O % Alcohol Reagent alcohol % Alcohol Reagent alcohol % Alcohol Reagent alcohol 1 9 Chloroform/Xylene I Use Chloroform 1 10 Chloroform/Xylene II Use Chloroform 1 11 Wax I Infiltration 2 12 Wax II No more than 2 hours 4 Total Rotate to the first container by pressing the rotation button. 7. Take sample holder out of oven, put samples into the holder, then place the holder into the holes on top of the 1 st container. 8. Make sure you are on Program 2, check by pressing program. Make sure the times are correct by pressing the clock button and +/- to change the time. 9. Program 2 is set to remain on container 12 for 4 hours, so stop after 2 hours. (To Stop, press Stop twice, turn agitation off, turn vacuum off). 10. To delay start: - Press Start, edit time. - Ex. START 0_18:00, 0 is number of days, 18:00 is time to start at. So this is set to start at 6 pm today. - Make sure to delay the right amount of time as the samples need to be taken out of 12 th container as soon as possible after 2 hours. Total preparation time: 2 hours if chemicals already diluted. 3 hours if not Embedding using embedding center machine 1. It will still be running after 14 hours as program 2 is currently set to remain in container 12 for 4 hours. To stop, press Stop twice, turn agitation off, turn vacuum off. 2. Press up arrow button to raise the holder. 3. Using a paper towel, ensure no wax falls on to the floor, and transfer the cassettes into the hot wax bath in the embedding center. This must be done as soon as possible as wax solidifies. 4. Cycle through the Cycle button until Cold Plate is seen. Press Set On Activate 5. Label peel-off molds according to the samples. 113

131 6. Fill the mold to the top with Wax by using the push-to-fill tap. 7. Take samples out of cassettes and place them into the mold filled with wax. 8. Orient the sample so that the sample is on its flat side and that the joint is facing upwards 9. Repeat with other samples. 10. Put it on the cold plate for 2 hours. 11. After 2 hours, turn off the cold plate by cycling to cold plate then pressing Set off Set 12. Put the blocks in a ziplock bag. Take as much of air out of the bag as possible. Total time: 1~2 hours Cutting the blocks (Use two microtomes if available) 1. Clean hot water bath glass. 2. Check the knob on water bath. It should be at 8, which corresponds to 50 degrees Celsius. Turn it on. 3. Fill glass with distilled water until it is half full Fill the other half with distilled water that has been heated in the microwave for 5 minutes, so the container is close to full. 4. Need #3 forceps, paint brush. 5. Turn on hot plate to 5~6. Get rid of air bubbles as much as you can. 6. Retract holder by pressing and holding up arrow. 7. Lock the block in place with tissue facing forward. Too much tightening breaks the block 8. Thickness setting: 8µm When practicing, practice using 10 µm. Once good enough, move down to 8µm. 9. Trim the block. Make sure the tissue remains untouched. The goal of trimming is to maximize the number of slices per glass slide (Max 5 per slide). 10. HP35N blade needed for bone cutting. However, it is no longer in production. Use A35 blade instead, which is a shorter blade. Place the blade into the blade holder and lock it in place. 11. Make sure the block surface is parallel to the blade. If not, adjust angle for both left-right and top-bottom. 12. Start cutting by rotating the wheel. Must turn the wheel clockwise. 13. Cut 8 and carefully move the cut slices into the water bath. Put wet gauze on the surface of the block to keep it from drying. 14. Transfer the slices onto the superfrost slides, and then place the slides onto the slide tray. The slide tray should be placed on the hot plate. 15. Time to time, observe the slices under the microscope to see how far the tissue has been cut. 16. Repeat 13~16 until the end of tissue is reached. 17. Sites of interest are from one end of full menisci to another, on both condyles. In between the condyles (ACL/PCL) can be skipped. 18. Wash hands after completion. Total time: 2 hours per 2 blocks 114

132 Staining (Protocol #12) New 2014 Table D.2 Table outlining the type of chemicals and time spent in each container of the automated tissue staining machine. Station Solution Time Exact 1 Xylene (can be old) 5 min. No 2 Xylene (can be old) 5 min. No 3 Xylene (Fresh) 5 min. No 4 100% Ethanol 1 min. No 5 100% Ethanol 1 min. No 6 50% Ethanol (dh 2 O) 2 min. No WASH1 Tap water 2 min. No 7 Weigert s Iron Hematoxylin 3 min. Yes WASH2 Tap water 2 min. No 8 Acid Alcohol (Differentiation 15 seconds Yes solution) WASH3 Tap water 2 min. 30 s No % Fast Green (2g in 1L dh 2 O, 3 min. Yes filter) 12 1% Acetic Acid (dh 2 O) 1 Dunk (1 second) Yes WASH4 Tap water 1 Dunk (1 second) No % Safranine-O (1g in 1L dh 2 O, 2 min. Yes filter) % Ethanol 1 min. 30 s No % Ethanol 1 min. 30 s No 18 Xylene 10s No Exit Xylene No - 100% xylene and alcohol do not affect the staining (staining chemicals are not soluble in it). Diluted alcohol will affect it due to water - Always mix with distilled water - Before staining the real deals, stain practice ones or the ones away from middle of the condyles to ensure the staining comes out the way you want. Do this every time you make a new batch of chemicals. - Weigert s hematoxylin loses its potency within 2-8 days of mixing. It depends how often it is used. Watch for the staining of nuclei. If it starts to stain any lighter, change. - We went through 1510 slides in 5 days, and it was still good. - Dilute all chemicals in distilled water - 1% = 1g/100mL 1. Switch on the machine via red button on the right side. 2. Take lids off of the rectangular chemical bottles inside (only the ones you are using). 3. Replace chemicals with our own, according to the table above. 115

133 a. Do not throw out Hematoxylene. It may be re-used, given that it is filtered every session. Using a filter paper, pour back into the original bottle (or a new bottle), then fill the container with filtered hematoxylene. b. Do NOT clean the containers with water. If sediment on bottom, pour some of its own liquid, shake, then pour out into waste. 4. Power on the cover-slipper switch in front, black. 5. Take the lid off of the xylene bath inside the cover-slipper. The bath is contained within a compartment which is accessible from the front of the machine. 6. Fill small xylene bottle up to the top in cover-slipper purpose is to keep the gold part wet. 7. Fill mounting media (Cytoseal XYL) bottle (minimum 100mL, maximum 200mL). 8. Load the black rectangular slide rack in the slot on the left side of the machine. 9. When check bath appears on the screen, slightly pull and push in the bath. 10. Now prime should appear. Press the prime button to prime, and the machine would prime. When it goes quiet, press the prime again. The message should disappear. 11. If it displays, Disp. Position, move the dispenser to the staining position. 12. From the Staining machine, press F1 to stain. 13. Choose program (Program 12). 14. Press Start. 15. Put slides into the black staining racks, and then put it into the loading station. Press Load 16. When the first rack is about half way, load another rack. Maximum of 3 simultaneous racks is recommended. 17. When the staining is about to be finished, say at the second last container/station, go over to the cover-slipper and transfer the xylene-dispenser to the 2 nd position. 18. Refill the slide rack on the cover-slipper machine. 19. The rack should automatically transfer from the staining machine to the cover-slipping machine. However, this usually isn t the case as the machine was ill-designed. If this happens, don t panic. Open the clear plastic cover, reach for the rack and pull it out. If beeping does not stop, press the red glowing button on the top of the machine in between the staining and cover-slipping machine. Manually transfer the rack to the xylene bath in the cover-slipping machine. Press start. 20. Racks can be put in every 5 minutes, and the machine has to be watched over while staining due to frequent errors such as coverslip error. Notes: - After about 10 racks, try to change the chemicals: Throw out xylene 1, xylene 3 into 2, xylene 2 into 1. Throw out ethanol 4 and put 5 in 4. Replace last 2 ethanols. - If the machine beeps at any stage of staining, check if anything is wrong. If no visible problems, turn it off and restart. - If slide rack is not moving from the stainer to the cover-slipper, manually move it to the coverslipper xylene bath. - The slide rack holds 30 slides per rack, and it takes around 50 minutes per rack to stain. However, if you do 3~4 racks simultaneously, you can save time. To do this, you need to have a station that can delay the timing, say 10 minute oven stage. Once the staining 116

134 starts and the first rack moves into the oven station, put a second rack into the rack compartment, and press the hold button to tell the machine that there is another rack in line. Continue until you have around 3~4 racks in the machine at the same time. Too many at once may cause the machine to trip, possibly resulting in longer durations in certain chemicals. Talk with Ruth September 9 th, Xylenes and 100% alcohol does not dissolve the staining solutions. The first three xylenes take the wax off so that the tissue can be stained. Right now, based on program 12, the rack goes from station 1 to 2 to 18. By doing so, xylene can drip into other stations Solution: go in a straight line. Change the program so that it goes from station 1 to 2 to 3 etc. Every 2 days (20 racks or so), throw xylene 1 and move xylene 2 to 1 and 3 to 2. Fresh xylene in 3 - The next alcohol steps are important as you need to take all the xylene off the slides before going into hematoxylin. Hematoxylin has water and alcohol in it, and xylene is not miscible in alcohol or water. This is going to affect hematoxylin staining Solution: Have another 100% alcohol station. Currently, it is 2 minutes in 1 alcohol. You can split this time into two stations (ie. 1 min in both) Watch if this gets cloudly. It would probably be okay for a week, but if it becomes cloudy, change - Water after hematoxylin is to wash off the excess hematoxylin. - Acid alcohol can be used for a long time. It removes excess hematoxylin - Water after acid alcohol is to remove alcohol - Acetic acid after fast green need to be changed frequently (every day or every 10 racks) - Fast green and safranin o are probably okay, but change as needed. Bacteria grow in fast green. - Fast green and safranin o dissolves in water. So lower concentration of alcohol takes away the stain, but 100% and xylene don t. - You need to take off all alcohol prior to moving into the coverslipper because the mounting media is not miscible in alcohol, so the slides may be cloudy or have problems with long term storage. 117

135 Appendix E: MICROCT ANALYSIS Written by SungYong (Kevin) Shin Position 3 Position 4 Position 2 Position 1 Figure E.1 Micro-computed tomography image showing the four positions of joints in a single scan. Setup: 1. Log in as invitro (ask the technician for the password). 2. There will be 2 terminal windows. If you want to open a new one, do so by going to session manager applications tab DECterms 3. Type ur in the terminal (ex. $ ur). Enter. 4. Type dir in the terminal (ex. $ dir). Enter. 5. Type cd cl p c (cd clark pilot com) (ex. $ cd cl p c). Enter. 6. Enter. 7. Open measurement. 8. Press the Task button. Figure E.1 The task button. 118

136 9. Select code by pressing Select and selecting the position and contour you are about to draw. Contour regions of interest: Anterior/Posterier Lateral/Medial Menisci (4), Medial/Lateral Femoral/Tibial trabaeculae (4), Medial/Lateral Femoral/Tibial Subchondral bone (4). A) 1. CONTOUR: POSITION # SAVE GOBJ FILES Menisci B) 2. CONTOUR: POSITION # SAVE GOBJ FILES Medial/Lateral Femoral/Tibial trabaeculae C) 3. CONTOUR: POSITION # - ANALYZE Medial/Lateral Femoral/Tibial Subchondral bone 10. Press OK then close 3D evaluation window. 11. File Save GOBJ as select the one you are about to contour OK Contouring Menisci: 1. Load task # 1 for the correct position. 2. Find a slice where meniscus appears. Pick one from anterior/posterior medial/lateral. Find a slice where the picked meniscus is the biggest. Good for anterior medial/lateral Good for posterior medial/lateral Figure E.2 Micro-computed tomography images showing the slice with largest anterior or posterior menisci. 119

137 3. Approximate frame: a. Anterior medial: ~312 b. Anterior lateral: ~303 c. Posterior medial: ~318 d. Posterior lateral: ~ Press the Contour button. Figure E.3 The contour button. 5. Make sure the contour tool is selected. Figure E.4 The contour tool. 6. Draw a rough contour around the meniscus of interest CLOCKWISE. Figure E.5 Micro-computed tomography image showing the meniscus contour. 7. Set inner value of contouring at 300, outer value at Double click on main window which will then draw an accurate contour for you. 120

138 Figure E.6 Micro-computed tomography image showing the corrected contour. 9. If not accurate enough, adjust by clicking the editing tool. Draw clockwise to modify. 10. Figure E.7 The editing tool. 11. Select Forwards, then click Iterate Forwards 12. Stop when meniscus disappears. If too much is iterated, delete contours by pressing Remove on keyboard. 13. Check each slide and modify if necessary. 14. Go back to initial slide and select backwards, then click Iterate Backwards 15. Stop when meniscus disappears. If too much is iterated, delete contours by pressing Remove on keyboard. 16. Check each slide and modify if necessary. 17. File Save GOBJ as Click the meniscus you contoured, then ok. 18. In the Contouring window, select all then delete all contours. 19. Repeat for other for menisci and samples. Contouring Medial/Lateral Femoral trabaeculae 1. Load task #2 for correct position. 2. Find a slide where growth plate is not apparent in the trabeculae. 121

139 Growth Plate + Trabeculae of left femur Good slice to start contouring on lateral side Figure E.8 Micro-computed tomography images showing growth plates appearing as well as a slice to begin the contour. 3. Draw a contour around the inner bone. Figure E.9 Micro-computed tomography image showing the contour of femoral trabeculae. 4. Skip 10 slides and draw another contour. 5. In the Contouring window, select Range and click on Morph to contour the slides in between. Modify if necessary. 6. Find the slide where last Trabeculae appears, and contour. Exclude less dense areas. Less dense meaning lighter areas. 122

140 Figure E.10 Micro-computed tomography image showing where the last femoral trabeculae appear. 7. Fill in between via contouring and morphing. Modify where necessary. 8. File Save GOBJ as Click the region you contoured, then ok. 9. In the Contouring window, select all then delete all contours. 10. Repeat for other for other samples. Contouring Medial/Lateral Tibial trabaeculae 1. Load task #2 for correct position. 2. Find a slide where Trabeculae first appear and draw contour inside the bone. Exclude less dense areas. Figure E.11 Micro-computed tomography image showing where the first tibial trabeculae appear. 3. Draw 2 contours when medial and lateral trabeculae are not joined, then morph. Modify contours if necessary. 123

141 Figure E.12 Micro-computed tomography images showing the contouring of tibial trabeculae. 4. Find the first slide where the medial and lateral trabeculae are joined, and draw a single contour. Exclude the area painted in red as it does not look like trabeculae. Figure E.13 Micro-computed tomography images showing how to modify the tibial trabeculae. 5. Find the first slide where the area with red paint in #4 disappear, and contour. Morph in between and modify if necessary. 6. Find the first slide that has growth plate appearing in either side of the tibia. If you see growth plate first appear in lateral (painted in red in the second figure), contour only the medial side. ` 124

142 Slide before growth plate appear in lateral side Only medial compartment contoured Figure E.14 Micro-computed tomography images showing how to contour near the growth plate (green). The growth plate appearing is marked in red. 7. Find the first slide that has growth plate appearing in the side you contoured in #6. Go to one slide before, contour, than morph. Modify if necessary. Slide with growth plate on medial side Slide just before Figure E.15 Micro-computed tomography images showing where growth plates appear and where to contour. 8. File Save GOBJ as Click the region you contoured, then ok. 9. In the Contouring window, select all then delete all contours. 10. Repeat for other for other samples. 125

143 Contouring Medial/Lateral Tibial trabaeculae BOX 1. Load task #2 for correct position. 2. Find the first slide where trabeculae hole first appears on the MEDIAL side. Go to slide just before that and contour a box around the medial side of the tibia. Make it as rectangular as possible (write the slide range from the Contouring Medial/Lateral Tibial trabaeculae and go to one slide before the range begins). Figure E.16 Micro-computed tomography image showing how to contour the trabeculae box to separate the medial and lateral compartment. 3. Copy the rectangular box by pressing Remove then replace it back by pressing Insert 4. Find the slide that you have selected as last slide when contouring tibial trabeculae, and Insert. Modify the rectangle so that it correctly separates medial and lateral. 126

144 Figure E.17 Micro-computed tomography image showing how to contour the trabeculae box to separate the medial and lateral compartment. 5. Morph in between. If it does not correctly separate medial and lateral, move the rectangle on the first or last slide by dragging with the left mouse click, then morph again. 6. File Save GOBJ as Click the region you contoured, then ok. 7. In the Contouring window, select all then delete all contours. 8. Repeat for other for other samples. Contouring Medial/Lateral Femoral/Tibial Subchondral bone 1. Load task #3 for correct position. 2. Find the slide where subchondral bone first appears and contour. Double click to get an accurate contour. (Lateral tibial plateau subchondral bone in this example). Figure E.18 Micro-computed tomography image showing the contouring of the first slice containing the tibial subchondral bone. 127

145 3. Count 12 slides including the first one deeper in to the bone. Contour that. Double click to get an accurate contour. Figure E.19 Micro-computed tomography image showing the contouring of the 12 th slice containing the tibial subchondral bone. 4. Morph in between and modify if necessary. 5. File Save GOBJ as Click the region you contoured, then ok. 6. In the Contouring window, select all then delete all contours. 7. Repeat with other subchondral bones. 128

146 Medial tibial plateau subchondral bone Lateral femoral condyle subchondral bone Medial femoral condyle subchondral bone Figure E.20 Micro-computed tomography image showing the contouring of the 1 st and 12 th slice containing the respective subchondral bone. Accessing 3D rendered images (After logging in as invitro): 1. type: a. ur b. uct_3d 2. Close the pop-up open file screen. Then go to read-clark-find the file you want. (ex. pilot project is located in DISK5:[BONELAB.PROJECTS.CLARK.PILOT.MODELS]*.AIM) 3. Nothing will change. Click on the blue screen once. 4. To change the orientation/zoom level etc, you have to wait until it renders completely (scale bar shows at the bottom) or manually stop it by click on the blue screen once again. 129

147 5. Once stopped, click and drag the blue screen to change orientation. You can also change different variables on the left menu. (Zoom is "scale factor). 6. If you don't see a scale bar, then the image is still being rendered. Either click on the image to stop rendering or wait until a scale bar appears. Adjusting Perspective 1. Use Subdim to adjust perspective. Depending on where the sample was during the initial scan, the x, y, and z axis differ. Play around with it to see how the image change and get to the region of interest. 2. Again, to use this, you have to stop the rendering. If you don't see a scale bar, then the image is still being rendered. Either click on the image to stop rendering or wait until a scale bar appears. Thickness Measurement 1. Open the wanted sample's 3D image. Thickness files are labelled TH at the end. 2. Go to the region of interest where you want to measure thickness. 3. To measure subchondral thickness, press shift while moving mouse to the region of measurement. Adjusting Color for different regions (femur, tibia, menisci, etc) 1. Open the wanted sample's 3D image. Thickness files are labelled TH at the end. 2. Stop rendering. 3. From menu, click Options --> Object Properties 130

148 Figure E.21 The object properties window. 4. From here, adjust from and to in order to define the region to change color. You have to experiment with this. 5. Change color and press ok. If it doesn't change what you want, try again. 131

149 Appendix F: MICROCT THICKNESS ANALYSIS Written by SungYong (Kevin) Shin Setup: 1. Log in as invitro (Ask the technician for password) Accessing 3D rendered images (After logging in as invitro): 1. type: a. ur b. uct_3d 2. Close the pop-up open file screen. Then go to read-clark-find the file you want. (ex. pilot project is located in DISK5:[BONELAB.PROJECTS.CLARK.DMM2.MODELS]*.AIM) 3. Nothing will change. Click on the blue screen once. 4. To change the orientation/zoom level etc, you have to wait until it renders completely (scale bar shows at the bottom) or manually stop it by click on the blue screen once again. 5. Once stopped, click and drag the blue screen to change orientation. You can also change different variables on the left menu. (Zoom is "scale factor). 6. If you don't see a scale bar, then the image is still being rendered. Either click on the image to stop rendering or wait until a scale bar appears. Adjusting Perspective 1. Use Subdim to adjust perspective. Depending on where the sample was during the initial scan, the x, y, and z axis differ. Play around with it to see how the image change and get to the region of interest. 2. Again, to use this, you have to stop the rendering. If you don't see a scale bar, then the image is still being rendered. Either click on the image to stop rendering or wait until a scale bar appears. 132

150 Thickness Measurement 1. Open the wanted sample's 3D image. Thickness files are labelled TH at the end. 2. Go to the region of interest where you want to measure thickness. For the purpose of this study, it will be the coronal section where both medial and lateral joint space is minimal. (i.e. Center of the contact region at normal physiological joint angle). 3. Measure the length of the condyle by pressing shift and clicking from where you want to measure and letting go where you want to stop measuring. Figure F.1 Micro-computed tomography model showing the measurement of a medial femoral condyle. 4. Find the position that corresponds to 25% of this length from one side of the condyle (i.e mm in this example). 133

151 Figure F.2 Micro-computed tomography model showing the measurement of the 25% of the measured length of the femoral condyle. 5. Then press shift and measure the thickness at that position. Figure F.3 Micro-computed tomography model showing the measurement of the thickness at the 25% of the measured length of the femoral condyle. 6. Repeat steps 4-5 at 50% and 70% of the measured condyle length. 7. Use a spreadsheet to record and calculate the positions and thickness. 134